CA2975449A1 - Method of production of gonadotrophin - Google Patents
Method of production of gonadotrophin Download PDFInfo
- Publication number
- CA2975449A1 CA2975449A1 CA2975449A CA2975449A CA2975449A1 CA 2975449 A1 CA2975449 A1 CA 2975449A1 CA 2975449 A CA2975449 A CA 2975449A CA 2975449 A CA2975449 A CA 2975449A CA 2975449 A1 CA2975449 A1 CA 2975449A1
- Authority
- CA
- Canada
- Prior art keywords
- sequence
- hcg
- cell
- seq
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 14
- 238000000034 method Methods 0.000 title description 49
- 150000007523 nucleic acids Chemical group 0.000 claims description 66
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 32
- 108020004707 nucleic acids Proteins 0.000 claims description 21
- 102000039446 nucleic acids Human genes 0.000 claims description 21
- 239000002299 complementary DNA Substances 0.000 claims description 7
- 108091033319 polynucleotide Proteins 0.000 claims description 7
- 102000040430 polynucleotide Human genes 0.000 claims description 7
- 239000002157 polynucleotide Substances 0.000 claims description 7
- 238000003306 harvesting Methods 0.000 claims description 6
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 101100468640 Danio rerio rhcgl2 gene Proteins 0.000 claims 1
- 101150053759 rhcg gene Proteins 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 136
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 41
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 41
- 230000009450 sialylation Effects 0.000 description 38
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 34
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 34
- 229940028334 follicle stimulating hormone Drugs 0.000 description 34
- 108090000623 proteins and genes Proteins 0.000 description 30
- 238000002360 preparation method Methods 0.000 description 25
- 235000018102 proteins Nutrition 0.000 description 23
- 102000004169 proteins and genes Human genes 0.000 description 23
- 239000000203 mixture Substances 0.000 description 21
- 239000000047 product Substances 0.000 description 20
- 239000002609 medium Substances 0.000 description 18
- 102000001708 Protein Isoforms Human genes 0.000 description 15
- 108010029485 Protein Isoforms Proteins 0.000 description 15
- 150000001413 amino acids Chemical class 0.000 description 15
- 210000005260 human cell Anatomy 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 238000001890 transfection Methods 0.000 description 13
- 210000002700 urine Anatomy 0.000 description 13
- 239000008194 pharmaceutical composition Substances 0.000 description 12
- 102100031974 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Human genes 0.000 description 11
- 108090000141 Sialyltransferases Proteins 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 101000703754 Homo sapiens CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Proteins 0.000 description 9
- 102000003838 Sialyltransferases Human genes 0.000 description 9
- 238000004113 cell culture Methods 0.000 description 9
- 229920001184 polypeptide Polymers 0.000 description 9
- 125000005629 sialic acid group Chemical group 0.000 description 8
- 230000002485 urinary effect Effects 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 230000002378 acidificating effect Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 229930182830 galactose Natural products 0.000 description 7
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 7
- 150000004676 glycans Chemical class 0.000 description 7
- 230000036512 infertility Effects 0.000 description 7
- 208000000509 infertility Diseases 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 108091026890 Coding region Proteins 0.000 description 6
- 150000001720 carbohydrates Chemical group 0.000 description 6
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 239000012228 culture supernatant Substances 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 231100000535 infertility Toxicity 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 229940024606 amino acid Drugs 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 235000000346 sugar Nutrition 0.000 description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 4
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 4
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 4
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 230000013595 glycosylation Effects 0.000 description 4
- 238000006206 glycosylation reaction Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 239000007972 injectable composition Substances 0.000 description 4
- 239000002523 lectin Substances 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 101710083574 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Proteins 0.000 description 3
- 108700010070 Codon Usage Proteins 0.000 description 3
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- -1 and the like) Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 230000005684 electric field Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000002826 placenta Anatomy 0.000 description 3
- 230000035935 pregnancy Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 2
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 101001054334 Homo sapiens Interferon beta Proteins 0.000 description 2
- 102000003996 Interferon-beta Human genes 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 108090001090 Lectins Proteins 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 102000002067 Protein Subunits Human genes 0.000 description 2
- 108010001267 Protein Subunits Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101150092923 St3gal4 gene Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 229920002988 biodegradable polymer Polymers 0.000 description 2
- 239000004621 biodegradable polymer Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 229960002442 glucosamine Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 229940099717 novarel Drugs 0.000 description 2
- 230000016087 ovulation Effects 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 230000003169 placental effect Effects 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 239000012521 purified sample Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- HORYNJCIACQHGZ-BHVWUGLYSA-N 1-[(3R,4R,5S,6R)-3-amino-2,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]ethanone Chemical compound C(C)(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO HORYNJCIACQHGZ-BHVWUGLYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 102000000827 Anterior Pituitary Hormones Human genes 0.000 description 1
- 108010001897 Anterior Pituitary Hormones Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101100263837 Bovine ephemeral fever virus (strain BB7721) beta gene Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 101100316840 Enterobacteria phage P4 Beta gene Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Polymers OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 description 1
- 102000003864 Human Follicle Stimulating Hormone Human genes 0.000 description 1
- 102000015611 Hypothalamic Hormones Human genes 0.000 description 1
- 108010024118 Hypothalamic Hormones Proteins 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 206010036590 Premature baby Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000003329 adenohypophysis hormone Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000002513 anti-ovulatory effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 108010064886 beta-D-galactoside alpha 2-6-sialyltransferase Proteins 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 238000011965 cell line development Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 229940015047 chorionic gonadotropin Drugs 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- HQPMKSGTIOYHJT-UHFFFAOYSA-N ethane-1,2-diol;propane-1,2-diol Chemical compound OCCO.CC(O)CO HQPMKSGTIOYHJT-UHFFFAOYSA-N 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010200 folin Substances 0.000 description 1
- 230000008217 follicular development Effects 0.000 description 1
- 108010006578 follitropin alfa Proteins 0.000 description 1
- 229960005210 follitropin alfa Drugs 0.000 description 1
- 108010081934 follitropin beta Proteins 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000033581 fucosylation Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 description 1
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
- 230000002710 gonadal effect Effects 0.000 description 1
- 229940057854 gonal f Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000601 hypothalamic hormone Substances 0.000 description 1
- 229940043650 hypothalamic hormone Drugs 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 230000000938 luteal effect Effects 0.000 description 1
- 230000001592 luteinising effect Effects 0.000 description 1
- 230000029849 luteinization Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000008063 pharmaceutical solvent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 229920001993 poloxamer 188 Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 229940071643 prefilled syringe Drugs 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000027380 protein glycosylation in Golgi Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000003206 sterilizing agent Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/99—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
- C12Y204/99004—Beta-galactoside alpha-2,3-sialyltransferase (2.4.99.4)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Endocrinology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Reproductive Health (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Diabetes (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
A host cell characterized in that it comprises integrated into its genome a sequence coding for the a chain of hCG, and use of the host cell to produce recombinant hCG.
Description
Method of production of gonadotrophin The present invention relates to gonadotrophins for use in the treatment of infertility. In particular it relates to human chorionic gonadotrophin (hCG).
The gonadotrophins are a group of heterodimeric glycoprotein hormones which regulate gonadal function in the male and female. They include follicle stimulating hormone (FSH), luteinising hormone (LH) and chorionic gonadotrophin (CG).
Human chorionic gonadotrophin (hCG) is secreted mainly by human placenta during pregnancy and supports the maintenance of corpus luteum.
hCG comprises a 92 amino acid alpha sub-unit (a chain), also common to the other glycoprotein hormones LH and FSH, and a 145 amino acid beta sub-unit ( [3 chain) unique to hCG, which dictates the hormone specificity. Each sub-unit is post translationally modified by the addition of complex carbohydrate residues.
The alpha sub-unit contains 2-N-linked glycosylation sites at amino acids 52 and 78 and the beta sub-unit contains 2-N-linked glycosylation sites at amino acids 13 and 30 and four 0-linked glycosylation sites at amino acids 121, 127, 132 and 138.
hCG extracted from the urine of pregnant women [Choragon (Ferring)]
has been used for many years in infertility treatment. The production of hCG
extracted from urine involves the collection and processing of large amounts of urine. Ovitrelle (Serono), a recombinant version of hCG, is also available for use in infertility treatment. This recombinant product is expressed in Chinese hamster ovary (CHO) cells, and has a different pharmacokinetic profile to hCG
produced from human urine.
There is considerable heterogeneity associated with hCG preparations which relates to differences in the amounts of various isoforms present.
Individual hCG isoforms exhibit identical amino acid sequences but differ in the extent to which they are post-translationally modified; particular isoforms are characterised by heterogeneity of the carbohydrate branch structures and differing amounts of sialic acid (a terminal sugar) incorporation, both of which appear to influence the specific isoform bioactivity.
The glycosylation of recombinant hCG ("rhCG") products reflects the range of glycosyl-transferases present in the host cell line. The existing rhCG
product, Ovitrelle, is derived from engineered Chinese hamster ovary cells (CHO
cells). The range of glycan modifications in CHO derived rhCG are more limited than those found on the natural products, derived from urine. Examples of the reduced glycan heterogeneity found in CHO derived rhCG include a lack of bisecting glucosamine and a reduced content of core fucosylation and acetyl lactosamine extensions. In addition, CHO cells are only able to add sialic acid using the a2,3 linkage (Kagawa eta!, 1988, Takeuchi eta!, 1988, Svensson et al., 1990).
Human urinary hCG (that is, hCG extracted from the urine of pregnant women) which has been used to date in treatment of infertility is, in actual fact, placental hCG; hCG produced in the (human) placenta and then excreted in the urine. This hCG is extracted from urine for use as the pharmaceutical product.
Bousfield et al (Rev Endocr Metab Disord (2011) 12:289-302) states that "while pituitary gonadotrophins possess both a2,3- and a2,6- linked sialic acid, placental hCG and recombinant gonadotrophins produced in Chinese hamster ovarian cells possess only a2,3- linked sialic acid". Thus, human urinary hCG (which is produced in the placenta) includes only a2,3- sialylation (like the CHO cell line derived recombinant products). Thus, the known pharmaceutical compositions which include hCG include only a2,3- linked sialic acid, but do not include a2,6-linked sialic acid.
It has been demonstrated that a recombinant FSH preparation (Organon) differs in the amounts of FSH with an isoelectric point (p1) of below 4 (considered the acidic isoforms) when compared to pituitary, serum or post-menopausal urine FSH (Ulloa-Aguirre et al. 1995). The amount of acidic isoforms in the urinary preparations of FSH was much higher as compared to the recombinant products, Gonal-f (Serono) and Puregon (Organon) (Andersen et al. 2004). This must reflect a lower molar content of sialic acid in rFSH since the content of negatively-charged glycan modified with sulphate is low in FSH. The lower sialic acid content, compared to natural FSH, is a feature of both commercially available FSH products and therefore must reflect a limitation in the manufacturing process (Bassett and Driebergen, 2005). The circulatory life-time of FSH has been documented for materials from a variety of sources. Some of these materials have been fractionated on the basis of overall molecular charge, as characterised by their pl, in which more acid equates to a higher negative charge. The major contributor to overall molecular charge is the total sialic content of each FSH
molecule. For instance, rFSH (Organon) has a sialic acid content of around 8 mol/mol, whereas urine-derived FSH has a higher sialic acid content (de Leeuw et al. 1996). The corresponding plasma clearance rates in the rat are 0.34 and 0.14 ml/min (Ulloa-Aguirre et al. 2003). In another example where a sample of recombinant FSH was split into high and low pl fractions, the in vivo potency of
The gonadotrophins are a group of heterodimeric glycoprotein hormones which regulate gonadal function in the male and female. They include follicle stimulating hormone (FSH), luteinising hormone (LH) and chorionic gonadotrophin (CG).
Human chorionic gonadotrophin (hCG) is secreted mainly by human placenta during pregnancy and supports the maintenance of corpus luteum.
hCG comprises a 92 amino acid alpha sub-unit (a chain), also common to the other glycoprotein hormones LH and FSH, and a 145 amino acid beta sub-unit ( [3 chain) unique to hCG, which dictates the hormone specificity. Each sub-unit is post translationally modified by the addition of complex carbohydrate residues.
The alpha sub-unit contains 2-N-linked glycosylation sites at amino acids 52 and 78 and the beta sub-unit contains 2-N-linked glycosylation sites at amino acids 13 and 30 and four 0-linked glycosylation sites at amino acids 121, 127, 132 and 138.
hCG extracted from the urine of pregnant women [Choragon (Ferring)]
has been used for many years in infertility treatment. The production of hCG
extracted from urine involves the collection and processing of large amounts of urine. Ovitrelle (Serono), a recombinant version of hCG, is also available for use in infertility treatment. This recombinant product is expressed in Chinese hamster ovary (CHO) cells, and has a different pharmacokinetic profile to hCG
produced from human urine.
There is considerable heterogeneity associated with hCG preparations which relates to differences in the amounts of various isoforms present.
Individual hCG isoforms exhibit identical amino acid sequences but differ in the extent to which they are post-translationally modified; particular isoforms are characterised by heterogeneity of the carbohydrate branch structures and differing amounts of sialic acid (a terminal sugar) incorporation, both of which appear to influence the specific isoform bioactivity.
The glycosylation of recombinant hCG ("rhCG") products reflects the range of glycosyl-transferases present in the host cell line. The existing rhCG
product, Ovitrelle, is derived from engineered Chinese hamster ovary cells (CHO
cells). The range of glycan modifications in CHO derived rhCG are more limited than those found on the natural products, derived from urine. Examples of the reduced glycan heterogeneity found in CHO derived rhCG include a lack of bisecting glucosamine and a reduced content of core fucosylation and acetyl lactosamine extensions. In addition, CHO cells are only able to add sialic acid using the a2,3 linkage (Kagawa eta!, 1988, Takeuchi eta!, 1988, Svensson et al., 1990).
Human urinary hCG (that is, hCG extracted from the urine of pregnant women) which has been used to date in treatment of infertility is, in actual fact, placental hCG; hCG produced in the (human) placenta and then excreted in the urine. This hCG is extracted from urine for use as the pharmaceutical product.
Bousfield et al (Rev Endocr Metab Disord (2011) 12:289-302) states that "while pituitary gonadotrophins possess both a2,3- and a2,6- linked sialic acid, placental hCG and recombinant gonadotrophins produced in Chinese hamster ovarian cells possess only a2,3- linked sialic acid". Thus, human urinary hCG (which is produced in the placenta) includes only a2,3- sialylation (like the CHO cell line derived recombinant products). Thus, the known pharmaceutical compositions which include hCG include only a2,3- linked sialic acid, but do not include a2,6-linked sialic acid.
It has been demonstrated that a recombinant FSH preparation (Organon) differs in the amounts of FSH with an isoelectric point (p1) of below 4 (considered the acidic isoforms) when compared to pituitary, serum or post-menopausal urine FSH (Ulloa-Aguirre et al. 1995). The amount of acidic isoforms in the urinary preparations of FSH was much higher as compared to the recombinant products, Gonal-f (Serono) and Puregon (Organon) (Andersen et al. 2004). This must reflect a lower molar content of sialic acid in rFSH since the content of negatively-charged glycan modified with sulphate is low in FSH. The lower sialic acid content, compared to natural FSH, is a feature of both commercially available FSH products and therefore must reflect a limitation in the manufacturing process (Bassett and Driebergen, 2005). The circulatory life-time of FSH has been documented for materials from a variety of sources. Some of these materials have been fractionated on the basis of overall molecular charge, as characterised by their pl, in which more acid equates to a higher negative charge. The major contributor to overall molecular charge is the total sialic content of each FSH
molecule. For instance, rFSH (Organon) has a sialic acid content of around 8 mol/mol, whereas urine-derived FSH has a higher sialic acid content (de Leeuw et al. 1996). The corresponding plasma clearance rates in the rat are 0.34 and 0.14 ml/min (Ulloa-Aguirre et al. 2003). In another example where a sample of recombinant FSH was split into high and low pl fractions, the in vivo potency of
2
3 the high pl (lower sialic acid content) fraction was decreased and it had a shorter plasma half-life (D'Antonio et al. 1999). The applicants have found that, similar to FSH, the known, CHO derived, recombinant hCG product (e.g. Ovitrelle) also has a lower amount of hCG with an isoelectric point (pp of below 4 (considered the acidic isoforms) than urinary hCG, also reflecting a lower sialic acid content of the known rhCG product compared to urinary hCG.
The applicants have developed a human derived recombinant hCG (that is, a recombinant hCG which is produced or expressed in a human cell line, e.g.
made by engineering a human cell line) which has a more acidic profile than the CHO cell line derived rhCG product, Ovitrelle, and which has a higher sialic acid content. This rhCG is the subject of International patent Application No.
PCT/GB2010/001854, published as WO 2011/042688. Recombinant hCG with a mixture of both a2,3 and a2,6-linked sialic acid was made by engineering a human cell line to express both rhCG and a2,3 sialyltransferase. The expressed product is highly acidic, having a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of for example 19.1 mol/mol, and carries a mix of both a2,3- and a2,6-linked sialic acids; the latter provided by the endogenous sialyl transferase activity. The applicants' research indicates that the type of sialic acid linkage, a2,3- or a2,6-, can have a dramatic influence on biological clearance of hCG. Human cell lines, as opposed to CHO cell lines, can express recombinant hCG with sialic acids attached by both a2,3 and a2,6 linkages.
The cell line of WO 2011/042688 expresses human derived recombinant hCG well. However, the cell line also expresses a relatively high level of the free 13 chain (free beta sub unit), and purification (to remove this free 13 chain from product hCG) was required.
The applicants have now developed a new cell line which expresses both rhCG and a2,3 sialyltransferase, and which produces rhCG with a reduced amount of the free 13 chain, thereby improving yield and reducing the level of product purification required (see Figure 7).
According to the present invention there is provided a (e.g. host) cell characterized in that it comprises integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 %
homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ
ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:4);
and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the sequence of SEQ ID NO:4). Preferably the (nucleic acid) sequence coding for the a chain of hCG is not the sequence which is banked as AH007338 (that is, preferably, the (nucleic acid) sequence coding for the a chain of hCG is not that shown in SEQ ID NO:5). The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise integrated into its genome a (nucleic acid) sequence coding for the 13 chain of hCG, for example the sequence according to SEQ ID
NO:2.
The host cell may be, for example, a PER.C6 cell, a HT1080 cell, a GT-5s cell etc.. Preferably the cell is a PER.C6 cell such as a PER.C6 cell deposited under ECACC no. 96022940.
According to the present invention there is provided a PER.C6 cell such as a PER.C6 cell deposited under ECACC no. 96022940, characterized in that it further comprises integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG. The (nucleic acid) sequence coding for the a chain of hCG may be selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 90 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ
ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98%
or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 90 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ ID NO:4); and a sequence which has at least 97 %
homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ
The applicants have developed a human derived recombinant hCG (that is, a recombinant hCG which is produced or expressed in a human cell line, e.g.
made by engineering a human cell line) which has a more acidic profile than the CHO cell line derived rhCG product, Ovitrelle, and which has a higher sialic acid content. This rhCG is the subject of International patent Application No.
PCT/GB2010/001854, published as WO 2011/042688. Recombinant hCG with a mixture of both a2,3 and a2,6-linked sialic acid was made by engineering a human cell line to express both rhCG and a2,3 sialyltransferase. The expressed product is highly acidic, having a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of for example 19.1 mol/mol, and carries a mix of both a2,3- and a2,6-linked sialic acids; the latter provided by the endogenous sialyl transferase activity. The applicants' research indicates that the type of sialic acid linkage, a2,3- or a2,6-, can have a dramatic influence on biological clearance of hCG. Human cell lines, as opposed to CHO cell lines, can express recombinant hCG with sialic acids attached by both a2,3 and a2,6 linkages.
The cell line of WO 2011/042688 expresses human derived recombinant hCG well. However, the cell line also expresses a relatively high level of the free 13 chain (free beta sub unit), and purification (to remove this free 13 chain from product hCG) was required.
The applicants have now developed a new cell line which expresses both rhCG and a2,3 sialyltransferase, and which produces rhCG with a reduced amount of the free 13 chain, thereby improving yield and reducing the level of product purification required (see Figure 7).
According to the present invention there is provided a (e.g. host) cell characterized in that it comprises integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 %
homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ
ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:4);
and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the sequence of SEQ ID NO:4). Preferably the (nucleic acid) sequence coding for the a chain of hCG is not the sequence which is banked as AH007338 (that is, preferably, the (nucleic acid) sequence coding for the a chain of hCG is not that shown in SEQ ID NO:5). The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise integrated into its genome a (nucleic acid) sequence coding for the 13 chain of hCG, for example the sequence according to SEQ ID
NO:2.
The host cell may be, for example, a PER.C6 cell, a HT1080 cell, a GT-5s cell etc.. Preferably the cell is a PER.C6 cell such as a PER.C6 cell deposited under ECACC no. 96022940.
According to the present invention there is provided a PER.C6 cell such as a PER.C6 cell deposited under ECACC no. 96022940, characterized in that it further comprises integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG. The (nucleic acid) sequence coding for the a chain of hCG may be selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 90 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ
ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98%
or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 90 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ ID NO:4); and a sequence which has at least 97 %
homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ
4 ID NO:4). Preferably the (nucleic acid) sequence coding for the a chain of hCG
is not the sequence which is banked as AH007338. The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise integrated into its genome a (nucleic acid) sequence coding for the 13 chain of hCG, for example the sequence according to SEQ ID NO:2.
It will be appreciated that the term "a sequence coding for the a chain of hCG" or "a (nucleic acid) sequence coding for the a chain of hCG" used herein also refers to a sequence coding for the a chain of FSH (or a sequence coding for the a chain of LH) because the a chain of hCG is the same as that for FSH (and LH).
The term "nucleic acid sequence" herein relates to any nucleic acid molecule that codes for polypeptides such as peptides, proteins etc. These nucleic acid molecules may be made of DNA, RNA or analogues thereof.
However, nucleic acid molecules being made of DNA are preferred.
The person skilled in the art is clearly aware that modification of a starting nucleotide sequence describes the process of optimization with respect to codon usage. The changes introduced can be easily identified by comparing the modified sequence and the starting sequence. Moreover, both sequences (that is, the starting sequence and the optimised sequence) will code for the same amino acid sequence.
The amino acid sequence of the a-chain of human hCG is disclosed in W02011/042688 (referred to as SEQ ID1 in that document), and is that described in Fiddes and Goodman 1979. It is banked under AH007388, and is shown as SEQ ID NO. 5 below. The amino acid sequence of the 13-chain of human hCG is depicted in SEQ ID No.2, and is banked as NP_000728. These amino acid sequences correspond to the wild-type amino acid sequences of the a- and the 13-chain of human hCG. The terms "wild-type nucleic acid sequence"
or "starting nucleic acid sequence" for the purposes of the present invention relate to a nucleic acid sequence which is intended to be used for (over)expression in a host cell and which has not been adapted to the codon usage in the host cell, but is the actual wild-type nucleic acid sequence coding for the protein. The term "optimized nucleic acid sequence" for the purposes of the present invention relates to a sequence that has been modified for expression in a host cell by adapting the sequence of the nonmodified/starting nucleic acid sequence. An optimized nucleic acid sequence codes for a protein having the same amino acid sequence as the protein encoded by the non-modified
is not the sequence which is banked as AH007338. The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise integrated into its genome a (nucleic acid) sequence coding for the 13 chain of hCG, for example the sequence according to SEQ ID NO:2.
It will be appreciated that the term "a sequence coding for the a chain of hCG" or "a (nucleic acid) sequence coding for the a chain of hCG" used herein also refers to a sequence coding for the a chain of FSH (or a sequence coding for the a chain of LH) because the a chain of hCG is the same as that for FSH (and LH).
The term "nucleic acid sequence" herein relates to any nucleic acid molecule that codes for polypeptides such as peptides, proteins etc. These nucleic acid molecules may be made of DNA, RNA or analogues thereof.
However, nucleic acid molecules being made of DNA are preferred.
The person skilled in the art is clearly aware that modification of a starting nucleotide sequence describes the process of optimization with respect to codon usage. The changes introduced can be easily identified by comparing the modified sequence and the starting sequence. Moreover, both sequences (that is, the starting sequence and the optimised sequence) will code for the same amino acid sequence.
The amino acid sequence of the a-chain of human hCG is disclosed in W02011/042688 (referred to as SEQ ID1 in that document), and is that described in Fiddes and Goodman 1979. It is banked under AH007388, and is shown as SEQ ID NO. 5 below. The amino acid sequence of the 13-chain of human hCG is depicted in SEQ ID No.2, and is banked as NP_000728. These amino acid sequences correspond to the wild-type amino acid sequences of the a- and the 13-chain of human hCG. The terms "wild-type nucleic acid sequence"
or "starting nucleic acid sequence" for the purposes of the present invention relate to a nucleic acid sequence which is intended to be used for (over)expression in a host cell and which has not been adapted to the codon usage in the host cell, but is the actual wild-type nucleic acid sequence coding for the protein. The term "optimized nucleic acid sequence" for the purposes of the present invention relates to a sequence that has been modified for expression in a host cell by adapting the sequence of the nonmodified/starting nucleic acid sequence. An optimized nucleic acid sequence codes for a protein having the same amino acid sequence as the protein encoded by the non-modified
5 sequence. The applicants have developed a optimised sequences which code for the a-chain of hCG (see e.g. SEQ ID No. 1 and 4, Figure 1).
According to the present invention in a further aspect there is provided a polynucleotide sequence (e.g. a polynucleotide sequence coding for the a chain of hCG) comprising a sequence selected from a nucleic acid sequence according to SEQ ID NO: 1; a nucleic acid sequence according to SEQ ID NO: 4; a nucleic acid sequence with at least 97% homology with (e.g. at least 98% or at least 99%
homology with) the nucleic acid of SEQ ID NO: 1 (e.g. a nucleic acid sequence with at least 97% sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the nucleic acid of SEQ ID NO: 1); and a nucleic acid sequence with at least 97% homology with (e.g. at least 98% or at least 99%
homology with) the nucleic acid of SEQ ID NO: 4 (e.g. a nucleic acid sequence with at least 97% sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the nucleic acid of SEQ ID NO: 4).
According to the present invention in a further aspect there is provided a method for producing recombinant hCG in a cell, comprising culturing the cell in a suitable medium and harvesting the recombinant protein (hCG) from said cell and/or said medium (e.g. harvesting the recombinant hCG from the cell culture supernatant), wherein the cell (host cell) comprises integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 90 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 90 %
sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97% sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 90 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ
ID NO:4); and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:4).
Preferably the (nucleic acid) sequence coding for the a chain of hCG is not the sequence which is banked as AH007338. The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise
According to the present invention in a further aspect there is provided a polynucleotide sequence (e.g. a polynucleotide sequence coding for the a chain of hCG) comprising a sequence selected from a nucleic acid sequence according to SEQ ID NO: 1; a nucleic acid sequence according to SEQ ID NO: 4; a nucleic acid sequence with at least 97% homology with (e.g. at least 98% or at least 99%
homology with) the nucleic acid of SEQ ID NO: 1 (e.g. a nucleic acid sequence with at least 97% sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the nucleic acid of SEQ ID NO: 1); and a nucleic acid sequence with at least 97% homology with (e.g. at least 98% or at least 99%
homology with) the nucleic acid of SEQ ID NO: 4 (e.g. a nucleic acid sequence with at least 97% sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the nucleic acid of SEQ ID NO: 4).
According to the present invention in a further aspect there is provided a method for producing recombinant hCG in a cell, comprising culturing the cell in a suitable medium and harvesting the recombinant protein (hCG) from said cell and/or said medium (e.g. harvesting the recombinant hCG from the cell culture supernatant), wherein the cell (host cell) comprises integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 90 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 90 %
sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97% sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 90 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ
ID NO:4); and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:4).
Preferably the (nucleic acid) sequence coding for the a chain of hCG is not the sequence which is banked as AH007338. The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise
6 integrated into its genome a (nucleic acid) sequence coding for the 13 chain of hCG, for example the sequence according to SEQ ID NO:2. The method may include a further step or steps of purifying the recombinant hCG obtained from said cell and/or said medium (e.g. purifying the recombinant hCG from the cell culture supernatant).
The (host) cell may be, for example, a PER.C6 cell, a HT1080 cell, a GT-5s cell etc.. Preferably the cell is a PER.C6 cell such as a PER.C6 cell deposited under ECACC no. 96022940.
According to the present invention in a further aspect there is provided a method for producing recombinant hCG in a cell, comprising culturing the cell in a suitable medium and harvesting the recombinant protein (hCG) from said cell and/or said medium (e.g. harvesting the recombinant hCG from the cell culture supernatant), wherein the cell is a PER.C6 cell (such as a PER.C6 cell deposited under ECACC no. 96022940) comprising integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG. The (nucleic acid) sequence coding for the a chain of hCG may be selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 90 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 90 %
sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 90 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ
ID NO:4); and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:4). Preferably the (nucleic acid) sequence coding for the a chain of hCG is not the sequence which is banked as AH007338. The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise integrated into its genome a (nucleic acid) sequence coding for the 13 chain of hCG, for example the sequence according to SEQ ID NO:2. The method may include a further step or steps of purifying the recombinant hCG obtained from said cell and/or said medium (e.g. purifying the recombinant hCG from the cell culture
The (host) cell may be, for example, a PER.C6 cell, a HT1080 cell, a GT-5s cell etc.. Preferably the cell is a PER.C6 cell such as a PER.C6 cell deposited under ECACC no. 96022940.
According to the present invention in a further aspect there is provided a method for producing recombinant hCG in a cell, comprising culturing the cell in a suitable medium and harvesting the recombinant protein (hCG) from said cell and/or said medium (e.g. harvesting the recombinant hCG from the cell culture supernatant), wherein the cell is a PER.C6 cell (such as a PER.C6 cell deposited under ECACC no. 96022940) comprising integrated into its genome a (nucleic acid) sequence coding for the a chain of hCG. The (nucleic acid) sequence coding for the a chain of hCG may be selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 90 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 90 %
sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID NO. 4; a sequence which has at least 90 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 90 % sequence identity with the sequence of SEQ
ID NO:4); and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:4). Preferably the (nucleic acid) sequence coding for the a chain of hCG is not the sequence which is banked as AH007338. The cell may further comprise integrated into its genome a cDNA encoding an alpha-2,3-sialyltransferase, for example a sequence according to SEQ ID NO: 3. The cell may further comprise integrated into its genome a (nucleic acid) sequence coding for the 13 chain of hCG, for example the sequence according to SEQ ID NO:2. The method may include a further step or steps of purifying the recombinant hCG obtained from said cell and/or said medium (e.g. purifying the recombinant hCG from the cell culture
7 supernatant).
The term "host cell" for the purposes of the present invention refers to any cell that is commonly used for expression, i.e. transcription and translation of nucleic acid sequences for the production of e.g. polypeptides. In particular, the term "host cell" or "organism" relates to prokaryotes, lower eukaryotes, plants, insect cells or mammalian cell culture systems. Preferably, the host cell is a mammalian cell, more preferably the host cell is a human cell, even more preferably a PERC6 cell.
The term "recombinant nucleic acid molecule" within the meaning of the present invention is intended to comprise all kinds of nucleic acid molecules which are capable of being introduced into a host cell and effecting the expression of a nucleic acid sequence which is contained within the recombinant nucleic acid molecule. The term includes, for example, plasmid vectors and viral vectors (e.g. adenoviral, lentiviral and retroviral vectors), as are well known in the art.
According to the present invention in a further aspect there is provided a recombinant nucleic acid molecule comprising a (first) nucleic acid sequence coding for the a chain of hCG, which (first) nucleic acid sequence is selected from a sequence according to SEQ ID NO: 1; a sequence which has at least 96.5 %
homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99%
homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID
NO. 4; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:14; and a sequence which has at least 97 %
homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ
ID NO:4). Preferably, the recombinant nucleic acid molecule of this aspect of the present invention comprises both an optimised nucleic acid sequence coding for the a-chain of human hCG and a nucleic acid sequence coding for the 13-chain of hCG. Preferably, the (first) nucleic acid sequence is under the control of a promoter which is active in a host cell. Preferably the recombinant nucleic acid molecule further comprises a second nucleic acid sequence coding for the 13
The term "host cell" for the purposes of the present invention refers to any cell that is commonly used for expression, i.e. transcription and translation of nucleic acid sequences for the production of e.g. polypeptides. In particular, the term "host cell" or "organism" relates to prokaryotes, lower eukaryotes, plants, insect cells or mammalian cell culture systems. Preferably, the host cell is a mammalian cell, more preferably the host cell is a human cell, even more preferably a PERC6 cell.
The term "recombinant nucleic acid molecule" within the meaning of the present invention is intended to comprise all kinds of nucleic acid molecules which are capable of being introduced into a host cell and effecting the expression of a nucleic acid sequence which is contained within the recombinant nucleic acid molecule. The term includes, for example, plasmid vectors and viral vectors (e.g. adenoviral, lentiviral and retroviral vectors), as are well known in the art.
According to the present invention in a further aspect there is provided a recombinant nucleic acid molecule comprising a (first) nucleic acid sequence coding for the a chain of hCG, which (first) nucleic acid sequence is selected from a sequence according to SEQ ID NO: 1; a sequence which has at least 96.5 %
homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99%
homology with) the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID NO:1); a sequence according to SEQ ID
NO. 4; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:14; and a sequence which has at least 97 %
homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ
ID NO:4). Preferably, the recombinant nucleic acid molecule of this aspect of the present invention comprises both an optimised nucleic acid sequence coding for the a-chain of human hCG and a nucleic acid sequence coding for the 13-chain of hCG. Preferably, the (first) nucleic acid sequence is under the control of a promoter which is active in a host cell. Preferably the recombinant nucleic acid molecule further comprises a second nucleic acid sequence coding for the 13
8 chain of hCG. The second nucleic acid sequence coding for the 13 chain of hCG
may be the sequence according to SEQ ID No. 2.
The second nucleic acid sequence may be under the control of a separate promoter which is active in a host cell. The first nucleic acid sequence and/or the second nucleic acid sequence may be under the control of a viral promoter (e.g. a CMVie promoter).
According to the present invention in a further aspect there is provided a host cell comprising or including a recombinant nucleic acid molecule as set out above (e.g. a host cell comprising a recombinant nucleic acid molecule comprising a (first) nucleic acid sequence coding for the a chain of hCG, which (first) nucleic acid sequence is selected from a sequence according to SEQ ID
NO: 1; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID
NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID
NO:1);
a sequence according to SEQ ID NO. 4; a sequence which has at least 96.5 %
homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:4); and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the sequence of SEQ ID NO:4).
The applicants have found that the cells (host cells), cell lines incorporating these host cells, recombinant nucleic acid molecules, polynucleotides and methods of the invention may produce recombinant hCG in high yield and purity (e.g. with little free beta chain).
According to the present invention in a further aspect there is provided a recombinant hCG ("rhCG" or "rechCG") which includes a2,3- and a2,6-sialylation, wherein the recombinant hCG is produced in a cell as described and claimed herein. The recombinant hCG may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of from 12 mol/mol to 20 mol/mol, for example from 12 mol/mol to 15.5 mol/mol, for example 12 mol/mol to 14.9 mol/mol. The rhCG (or rhCG preparation) may have a sialic acid content of 12.5 mol/mol to 14.5 mol/mol, for example a sialic acid content of 12.8 mol/mol to 13.2 mol/mol. The rhCG (or rhCG preparation) according to the
may be the sequence according to SEQ ID No. 2.
The second nucleic acid sequence may be under the control of a separate promoter which is active in a host cell. The first nucleic acid sequence and/or the second nucleic acid sequence may be under the control of a viral promoter (e.g. a CMVie promoter).
According to the present invention in a further aspect there is provided a host cell comprising or including a recombinant nucleic acid molecule as set out above (e.g. a host cell comprising a recombinant nucleic acid molecule comprising a (first) nucleic acid sequence coding for the a chain of hCG, which (first) nucleic acid sequence is selected from a sequence according to SEQ ID
NO: 1; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO:1 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:1); a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID
NO:1 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99% sequence identity with) the sequence of SEQ ID
NO:1);
a sequence according to SEQ ID NO. 4; a sequence which has at least 96.5 %
homology with the sequence of SEQ ID NO: 4 (e.g. a sequence which has at least 96.5 % sequence identity with the sequence of SEQ ID NO:4); and a sequence which has at least 97 % homology with (e.g. at least 98% or at least 99% homology with) the sequence of SEQ ID NO:4 (e.g. a sequence which has at least 97 % sequence identity with (e.g. at least 98% or at least 99%
sequence identity with) the sequence of SEQ ID NO:4).
The applicants have found that the cells (host cells), cell lines incorporating these host cells, recombinant nucleic acid molecules, polynucleotides and methods of the invention may produce recombinant hCG in high yield and purity (e.g. with little free beta chain).
According to the present invention in a further aspect there is provided a recombinant hCG ("rhCG" or "rechCG") which includes a2,3- and a2,6-sialylation, wherein the recombinant hCG is produced in a cell as described and claimed herein. The recombinant hCG may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of from 12 mol/mol to 20 mol/mol, for example from 12 mol/mol to 15.5 mol/mol, for example 12 mol/mol to 14.9 mol/mol. The rhCG (or rhCG preparation) may have a sialic acid content of 12.5 mol/mol to 14.5 mol/mol, for example a sialic acid content of 12.8 mol/mol to 13.2 mol/mol. The rhCG (or rhCG preparation) according to the
9 invention may have a sialic acid content of 13 mol/mol.
In examples of the invention, the rhCG may be present as a single isoform or as a mixture of isoforms.
The recombinant hCG (or rhCG preparation) produced by the methods and cells of the invention includes a2,3- and a2,6-sialylation. By sialylation it is meant the amount of sialic residues present on the hCG carbohydrate structures.
a2,3-sialylation means sialylation at the 2,3 position (as is well known in the art);
and a2,6 sialylation means sialylation at the 2,6 position (also well known in the art). Thus, herein, the wording "`"/0 of the total sialylation may be a 2,3 sialylation"
refers to the % of the total number of sialic acid residues present in the hCG
which are sialylated in the 2,3 position. The term "`"/0 of the total sialylation being a2,6-sialylation" refers to the % of the total number of sialic acid residues present in the hCG which are sialylated in the 2,6 position. The term "`"/0 of the total sialylation being a2,8-sialylation" refers to the % of the total number of sialic acid residues present in the hCG which are sialylated in the 2,8 position.
The rhCG (or rhCG preparation) produced by the methods and cells according to the invention may have 1% to 99% of the total sialylation being a2,3-sialylation. The rhCG (or rhCG preparation) produced by the methods and cells according to the invention may have 1% to 90% of the total sialylation being a2,3-sialylation. The rhCG (or rhCG preparation) according to the invention may have
In examples of the invention, the rhCG may be present as a single isoform or as a mixture of isoforms.
The recombinant hCG (or rhCG preparation) produced by the methods and cells of the invention includes a2,3- and a2,6-sialylation. By sialylation it is meant the amount of sialic residues present on the hCG carbohydrate structures.
a2,3-sialylation means sialylation at the 2,3 position (as is well known in the art);
and a2,6 sialylation means sialylation at the 2,6 position (also well known in the art). Thus, herein, the wording "`"/0 of the total sialylation may be a 2,3 sialylation"
refers to the % of the total number of sialic acid residues present in the hCG
which are sialylated in the 2,3 position. The term "`"/0 of the total sialylation being a2,6-sialylation" refers to the % of the total number of sialic acid residues present in the hCG which are sialylated in the 2,6 position. The term "`"/0 of the total sialylation being a2,8-sialylation" refers to the % of the total number of sialic acid residues present in the hCG which are sialylated in the 2,8 position.
The rhCG (or rhCG preparation) produced by the methods and cells according to the invention may have 1% to 99% of the total sialylation being a2,3-sialylation. The rhCG (or rhCG preparation) produced by the methods and cells according to the invention may have 1% to 90% of the total sialylation being a2,3-sialylation. The rhCG (or rhCG preparation) according to the invention may have
10% or more of the total sialylation being a2,3-sialylation, for example, 10%
to 90% of the total sialylation may be a2,3-sialylation. For example, 20, 30, 40, 45, 50, 55, 60, 70, 80 or 90% or more of the total sialylation may be a2,3-sialylation.
45% to 80% of the total sialylation may be a2,3-sialylation, for example 50%
to 70% of the total sialylation may be a2,3-sialylation, for example 55 to 65% of the total sialylation may be a2,3-sialylation. The rhCG (or rhCG preparation) may include a2,3-sialylation in an amount which is from 65 to 95% of the total sialylation, for example from 70 to 90% of the total sialylation, for example from 85 to 90% of the total sialylation.
The rhCG (or rhCG preparation) produced by the methods and cells of the invention may have 1 to 99% of the total sialylation being a2,6-sialylation.
The rhCG (or rhCG preparation) of the invention may have 5 to 50% of the total sialylation being a2,6-sialylation. For example 5 to 45%, for example 6 to 40%, for example 7 to 30%, for example 8 to 20% of the total sialylation may be a2,6-sialylation. The rhCG (or rhCG preparation) may include a2,6-sialylation in an amount which is from 20-75% of the total sialylation, for example, 30-60% of the total sialylation, for example 35-45% of the total sialylation. The rhCG (or rhCG
preparation) may include a2,6-sialylation in an amount which is from 5 to 35%
of the total sialylation, for example from 10 to 20% of the total sialylation.
For example 11-55% of the total sialylation may be a2,6- sialylation.
The rhCG or rhCG preparation produced by the methods and cells may optionally further include a2,8 sialylation. The rhCG (or rhCG preparation) of the invention may have 5% or less of the total sialylation being a2,8-sialylation, for example 0 to 4%, e.g. 0.1-4% of the total sialylation may be a2,8-sialylation. The rhCG (or rhCG preparation) of the invention may have no a2,8-sialylation.
The rhCG (or rhCG preparation) produced by the methods and cells according to the invention may have a sialic acid content (amount of sialylation per hCG molecule) of (based on the mass of protein, rather than the mass of protein plus carbohydrate) of 6% or greater (e.g. between 6% and 15%, e.g.
between 7% and 13%, e.g. between 8% and 12%, e.g. between 11% and 15%, e.g. between 12% and 14%) by mass.
The rhCG (or rhCG preparation) may be produced or expressed in a human cell line, for example a PER.C6 cell line, a HT1080 cell line etc.. The rhCG (or rhCG preparation) may be produced or expressed in a human derived cell line or a modified human cell line, for example a PER.C6 derived cell line or modified PER.C6 cell line, a modified HT1080 cell line or HT1080 derived cell line etc.. In a preferred example, the rhCG is produced or expressed in a PER.C6 cell line, a PER.C6 derived cell line or a modified PER.C6 cell line.
rhCG which is produced or expressed in a human cell line (e.g. a PER.C6 cell line, a HT1080 cell line, a GT-5s cell line) will include some a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line] and will include some a2,3-linked sialic acids (a2,3 sialylation) provided by endogenous sialyl transferase activity [of the cell line]. The cell line may be modified using a2,3-sialyltransferase. Alternatively or additionally, the cell line may be modified using a2,6-sialyltransferase. This may simplify (and render more efficient) the production method because manipulation and control of e.g.
the cell growth medium to retain sialylation may be less critical than with known processes. The method may also be more efficient because there is little basic rhCG produced compared to production of known rhCG products; more acidic rhCG is produced and separation/removal of basic hCG is less problematic.
The rhCG may be produced using an a2,3- sialyltransferase, for example produced using a human cell line modified using an a2,3-sialyltransferase. The rhCG may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity. The rhCG may be produced using an
to 90% of the total sialylation may be a2,3-sialylation. For example, 20, 30, 40, 45, 50, 55, 60, 70, 80 or 90% or more of the total sialylation may be a2,3-sialylation.
45% to 80% of the total sialylation may be a2,3-sialylation, for example 50%
to 70% of the total sialylation may be a2,3-sialylation, for example 55 to 65% of the total sialylation may be a2,3-sialylation. The rhCG (or rhCG preparation) may include a2,3-sialylation in an amount which is from 65 to 95% of the total sialylation, for example from 70 to 90% of the total sialylation, for example from 85 to 90% of the total sialylation.
The rhCG (or rhCG preparation) produced by the methods and cells of the invention may have 1 to 99% of the total sialylation being a2,6-sialylation.
The rhCG (or rhCG preparation) of the invention may have 5 to 50% of the total sialylation being a2,6-sialylation. For example 5 to 45%, for example 6 to 40%, for example 7 to 30%, for example 8 to 20% of the total sialylation may be a2,6-sialylation. The rhCG (or rhCG preparation) may include a2,6-sialylation in an amount which is from 20-75% of the total sialylation, for example, 30-60% of the total sialylation, for example 35-45% of the total sialylation. The rhCG (or rhCG
preparation) may include a2,6-sialylation in an amount which is from 5 to 35%
of the total sialylation, for example from 10 to 20% of the total sialylation.
For example 11-55% of the total sialylation may be a2,6- sialylation.
The rhCG or rhCG preparation produced by the methods and cells may optionally further include a2,8 sialylation. The rhCG (or rhCG preparation) of the invention may have 5% or less of the total sialylation being a2,8-sialylation, for example 0 to 4%, e.g. 0.1-4% of the total sialylation may be a2,8-sialylation. The rhCG (or rhCG preparation) of the invention may have no a2,8-sialylation.
The rhCG (or rhCG preparation) produced by the methods and cells according to the invention may have a sialic acid content (amount of sialylation per hCG molecule) of (based on the mass of protein, rather than the mass of protein plus carbohydrate) of 6% or greater (e.g. between 6% and 15%, e.g.
between 7% and 13%, e.g. between 8% and 12%, e.g. between 11% and 15%, e.g. between 12% and 14%) by mass.
The rhCG (or rhCG preparation) may be produced or expressed in a human cell line, for example a PER.C6 cell line, a HT1080 cell line etc.. The rhCG (or rhCG preparation) may be produced or expressed in a human derived cell line or a modified human cell line, for example a PER.C6 derived cell line or modified PER.C6 cell line, a modified HT1080 cell line or HT1080 derived cell line etc.. In a preferred example, the rhCG is produced or expressed in a PER.C6 cell line, a PER.C6 derived cell line or a modified PER.C6 cell line.
rhCG which is produced or expressed in a human cell line (e.g. a PER.C6 cell line, a HT1080 cell line, a GT-5s cell line) will include some a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line] and will include some a2,3-linked sialic acids (a2,3 sialylation) provided by endogenous sialyl transferase activity [of the cell line]. The cell line may be modified using a2,3-sialyltransferase. Alternatively or additionally, the cell line may be modified using a2,6-sialyltransferase. This may simplify (and render more efficient) the production method because manipulation and control of e.g.
the cell growth medium to retain sialylation may be less critical than with known processes. The method may also be more efficient because there is little basic rhCG produced compared to production of known rhCG products; more acidic rhCG is produced and separation/removal of basic hCG is less problematic.
The rhCG may be produced using an a2,3- sialyltransferase, for example produced using a human cell line modified using an a2,3-sialyltransferase. The rhCG may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity. The rhCG may be produced using an
11 a2,3- and/or an a2,6-sialyltransferase, for example produced using a human cell line modified using an a2,3-sialyltransferase and/or an a2,6-sialyltransferase.
According to the present invention in a further aspect there is provided a recombinant hCG or a recombinant hCG preparation as described herein which is produced or expressed by the methods described herein.
The rhCG structure contains glycan moieties. Branching can occur with the result that the glycan may have 1, 2, 3, 4 or more terminal sugar residues or "antennae", as is well known in the art. The rhCG according to aspects of the invention of the invention may have glycans with mono-antennary and/or di-antennary and/or tri-antennary and/or tetra-antennary glycan structures. The rhCG may include mono-antennary and/or bi-antennary and/or tri-antennary and/or tetra-antennary glycan structures, for example with relative amounts as follows: 0.1 to 3% mono-antennary; 65% to 85% bi-antennary; 15 to 25% tri-antennary and 0.5 to 1.5% tetra-antennary (e.g. as shown by WAX analysis of charged glycans).
According to the present invention in a further aspect there is provided a pharmaceutical composition comprising a recombinant hCG (rhCG) having a2,3-sialylation and a2,6-sialylation, wherein the recombinant hCG is produced by the methods and/or cells disclosed herein. The rhCG may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of from
According to the present invention in a further aspect there is provided a recombinant hCG or a recombinant hCG preparation as described herein which is produced or expressed by the methods described herein.
The rhCG structure contains glycan moieties. Branching can occur with the result that the glycan may have 1, 2, 3, 4 or more terminal sugar residues or "antennae", as is well known in the art. The rhCG according to aspects of the invention of the invention may have glycans with mono-antennary and/or di-antennary and/or tri-antennary and/or tetra-antennary glycan structures. The rhCG may include mono-antennary and/or bi-antennary and/or tri-antennary and/or tetra-antennary glycan structures, for example with relative amounts as follows: 0.1 to 3% mono-antennary; 65% to 85% bi-antennary; 15 to 25% tri-antennary and 0.5 to 1.5% tetra-antennary (e.g. as shown by WAX analysis of charged glycans).
According to the present invention in a further aspect there is provided a pharmaceutical composition comprising a recombinant hCG (rhCG) having a2,3-sialylation and a2,6-sialylation, wherein the recombinant hCG is produced by the methods and/or cells disclosed herein. The rhCG may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of from
12 mol/mol to 15.5 mol/mol, for example 12 mol/mol to 14.9 mol/mol (e.g. as set out above). The rhCG may have a sialic acid content of 12.5 mol/mol to 14.5 mol/mol, for example a sialic acid content of 12.8 mol/mol to 13.2 mol/mol.
Preferably the rhCG according to the invention has a sialic acid content of 13 mol/mol.
The pharmaceutical composition may further comprise FSH and/or LH.
FSH can be obtained by any means known in the art. FSH as used herein includes human-derived and recombinant FSH. Human-derived FSH can be purified from any appropriate source (e.g. urine) by any method known in the art.
The FSH may be recombinant FSH ¨ for example expressed in a human cell line.
Methods of expressing and purifying recombinant FSH are well known in the art.
LH can be obtained by any means known in the art. LH, as used herein, includes human-derived and recombinant LH. Human-derived LH can be purified from any appropriate source (e.g. urine) by any method known in the art.
Methods of expressing and purifying recombinant LH are known in the art.
The pharmaceutical composition may be for, or for use in, the treatment of infertility, e.g. for use in e.g. assisted reproductive technologies (ART), ovulation
Preferably the rhCG according to the invention has a sialic acid content of 13 mol/mol.
The pharmaceutical composition may further comprise FSH and/or LH.
FSH can be obtained by any means known in the art. FSH as used herein includes human-derived and recombinant FSH. Human-derived FSH can be purified from any appropriate source (e.g. urine) by any method known in the art.
The FSH may be recombinant FSH ¨ for example expressed in a human cell line.
Methods of expressing and purifying recombinant FSH are well known in the art.
LH can be obtained by any means known in the art. LH, as used herein, includes human-derived and recombinant LH. Human-derived LH can be purified from any appropriate source (e.g. urine) by any method known in the art.
Methods of expressing and purifying recombinant LH are known in the art.
The pharmaceutical composition may be for, or for use in, the treatment of infertility, e.g. for use in e.g. assisted reproductive technologies (ART), ovulation
13 PCT/EP2016/059006 induction or intrauterine insemination (IUD. The pharmaceutical composition may be for, or for use in, triggering final maturation / ovulation and luteinization, the stimulation of follicular development in women with severe LH and FSH
deficiency, and/or in luteal support (rhCG/rLH). The pharmaceutical composition may be for, or for use in, inducing monofollicular development in anovulatory WHO Type II women (e.g. rhCG with rLH), for enhancing multifollicular response with LH priming in patients undergoing COH (e.g. rhCG with rLH), and for supplementation to COH to improve implantation/pregnancy rates (e.g. rhCG with rLH), and/or increasing pregnancy rates in assisted reproductive technologies.
The pharmaceutical composition may be for, or for use in, the prevention of miscarriages, and/or prematurity prevention. The pharmaceutical composition may be for, or for use in, the treatment of endometriosis.
The pharmaceutical composition may be used, for example, in medical indications where known hCG preparations are used. The present invention also provides the use of rhCG and/or an rhCG preparation described herein (according to aspects of the invention) for, or in the manufacture of a medicament for, the treatment of infertility.
The pharmaceutical compositions of the present invention may be formulated into well-known compositions for any route of drug administration, e.g.
oral, rectal, parenteral, transdermal (e.g. patch technology), intravenous, intramuscular, subcutaneous, intracisternal, intravaginal, intraperitoneal, local (powders, ointments or drops) or as a buccal or nasal spray. A typical composition comprises a pharmaceutically acceptable carrier, such as aqueous solution, non toxic excipients, including salts and preservatives, buffers and the like, as described in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), at pages 1405 to 1412 and 1461 ¨ 87, and the national formulary XIV fourteenth edition (American Pharmaceutical Association, 1975), among others. Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectible organic esters such as ethyl oleate. The compositions of the present invention also can contain additives such as but not limited to preservatives, wetting agents, emulsifying agents, and dispersing agents.
Antibacterial and antifungal agents can be included to prevent growth of microbes and includes, for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like. Furthermore, it may be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
In some cases, to effect prolonged action it is desirable to slow the absorption of hCG (and other active ingredients, if present) from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility.
The rate of absorption of hCG then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered hCG combination form is accomplished by dissolving or suspending the hCG combination in an oil vehicle. Injectable depot forms can be made by forming microencapsule matrices of the hCG (and other agents, if present) in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of hCG to polymer and the nature of the particular polymer employed, the rate of hCG release can be controlled.
Examples of other biodegradable polymers include polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides) etc. Depot injectable formulations are also prepared by entrapping the hCG in liposomes or microemulsions which are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
Injectable formulations can be supplied in any suitable container, e.g. vial, pre-filled syringe, injection cartridges, and the like.
Formulations (e.g. injectable formulation can be supplied as a product having pharmaceutical compositions containing hCG (optionally with FSH, LH
etc.) If there is more than one active ingredient (i.e. hCG and e.g. FSH or LH) these may be suitable for administration separately or together. If administered separately, administration can be sequential. The product can be supplied in any appropriate package. For example, a product can contain a number of pre-filled syringes or vials containing either hCG, FSH, or a combination of both FSH and hCG. The syringes or vials may be packaged in a blister package or other means to maintain sterility. A product can optionally contain instructions for using the hCG and FSH formulations.
The pH and exact concentration of the various components of the pharmaceutical composition are adjusted in accordance with routine practice in this field. See GOODMAN and GILMAN's THE PHARMACOLOGICAL BASIS
FOR THERAPEUTICS, 71h ed. In a preferred embodiment, the compositions of the invention are supplied as compositions for parenteral administration.
General
deficiency, and/or in luteal support (rhCG/rLH). The pharmaceutical composition may be for, or for use in, inducing monofollicular development in anovulatory WHO Type II women (e.g. rhCG with rLH), for enhancing multifollicular response with LH priming in patients undergoing COH (e.g. rhCG with rLH), and for supplementation to COH to improve implantation/pregnancy rates (e.g. rhCG with rLH), and/or increasing pregnancy rates in assisted reproductive technologies.
The pharmaceutical composition may be for, or for use in, the prevention of miscarriages, and/or prematurity prevention. The pharmaceutical composition may be for, or for use in, the treatment of endometriosis.
The pharmaceutical composition may be used, for example, in medical indications where known hCG preparations are used. The present invention also provides the use of rhCG and/or an rhCG preparation described herein (according to aspects of the invention) for, or in the manufacture of a medicament for, the treatment of infertility.
The pharmaceutical compositions of the present invention may be formulated into well-known compositions for any route of drug administration, e.g.
oral, rectal, parenteral, transdermal (e.g. patch technology), intravenous, intramuscular, subcutaneous, intracisternal, intravaginal, intraperitoneal, local (powders, ointments or drops) or as a buccal or nasal spray. A typical composition comprises a pharmaceutically acceptable carrier, such as aqueous solution, non toxic excipients, including salts and preservatives, buffers and the like, as described in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), at pages 1405 to 1412 and 1461 ¨ 87, and the national formulary XIV fourteenth edition (American Pharmaceutical Association, 1975), among others. Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectible organic esters such as ethyl oleate. The compositions of the present invention also can contain additives such as but not limited to preservatives, wetting agents, emulsifying agents, and dispersing agents.
Antibacterial and antifungal agents can be included to prevent growth of microbes and includes, for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like. Furthermore, it may be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
In some cases, to effect prolonged action it is desirable to slow the absorption of hCG (and other active ingredients, if present) from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility.
The rate of absorption of hCG then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered hCG combination form is accomplished by dissolving or suspending the hCG combination in an oil vehicle. Injectable depot forms can be made by forming microencapsule matrices of the hCG (and other agents, if present) in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of hCG to polymer and the nature of the particular polymer employed, the rate of hCG release can be controlled.
Examples of other biodegradable polymers include polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides) etc. Depot injectable formulations are also prepared by entrapping the hCG in liposomes or microemulsions which are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
Injectable formulations can be supplied in any suitable container, e.g. vial, pre-filled syringe, injection cartridges, and the like.
Formulations (e.g. injectable formulation can be supplied as a product having pharmaceutical compositions containing hCG (optionally with FSH, LH
etc.) If there is more than one active ingredient (i.e. hCG and e.g. FSH or LH) these may be suitable for administration separately or together. If administered separately, administration can be sequential. The product can be supplied in any appropriate package. For example, a product can contain a number of pre-filled syringes or vials containing either hCG, FSH, or a combination of both FSH and hCG. The syringes or vials may be packaged in a blister package or other means to maintain sterility. A product can optionally contain instructions for using the hCG and FSH formulations.
The pH and exact concentration of the various components of the pharmaceutical composition are adjusted in accordance with routine practice in this field. See GOODMAN and GILMAN's THE PHARMACOLOGICAL BASIS
FOR THERAPEUTICS, 71h ed. In a preferred embodiment, the compositions of the invention are supplied as compositions for parenteral administration.
General
14 methods for the preparation of the parenteral formulations are known in the art and are described in REMINGTON; THE SCIENCE AND PRACTICE OF
PHARMACY, supra, at pages 780-820. The parenteral compositions can be supplied in liquid formulation or as a solid which will be mixed with a sterile injectable medium just prior to administration. In an embodiment, the parenteral compositions are supplied in dosage unit form for ease of administration and uniformity of dosage.
Detailed description of the invention The present invention will now be described in more detail with reference to the following Examples and to the attached drawings in which:
Figure 1 shows the nucleic acid and derived amino acid sequences of the optimised hCG alpha sequence;
Figure 2 shows the nucleic acid and derived amino acid sequences of hCG beta sequence;
Figure 3 shows the nucleic acid and derived amino acid sequences of ST3GAL4 Figure 4 shows a plasmid map of the phCGalpha/beta expression vector;
Figure 5 shows the a2,3-sialyltransferase (ST3GAL4) expression vector;
Figure 6 shows the detection of rhCG lsoforms by IEF stained with Coomassie Blue in compositions according to the invention (Lanes 4, 5, 6 and 7, 15 pg per lane); the CHO derived composition of the prior art, Ovitrelle (Lane 2, 15pg);
and a human urinary product obtained from pregnant women Novarel (Lane 3, 15pg);
and Figure 7 compares the concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced using the cells of the invention including the "Optimised" nucleic sequence of the alpha sub unit, compared with concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced in comparative example cells including the WT (wild-type) alpha sub unit;
Examples 1 to 7 Overview of cell line development process A single plasmid carrying hCG alpha and beta chains, each driven from a separate CMVie promoter, was transfected into PER.C60 cells by electroporation.
This plasmid carried a neomycin resistance gene cassette (Fig 4) allowing for selection of transfectants using G418 (Geneticin). Pools of transfectants were expanded and then subjected to limiting dilution cloning in 96 well plates under G418 selection. Clonal lines were expanded and high expressing colonies were identified based on hCG titre in the cell culture supernatant.
Five lead clones were selected, based on high productivity, and each was then transfected with a second plasmid expressing the a2,3-sialyltransferase gene ST3GAL4 along with the Hygromycin selection maker (Fig 5). Each of the five transfection pools was expanded and cell culture supernatants were assessed for hCG concentration and in vivo pharmacokinetics (PK).
Promising pools were then subjected to another round of limited dilution cloning. The resulting clones were expanded and cell culture supernatants were assessed for hCG concentration and exposed galactose on the hCG protein (by lectin ELISA). Those with a low level of exposed galactose were subjected to further assays including in vivo PK. Clones that produced hCG with optimum PK
profiles, had low exposed galactose and expressed the protein at high levels were assessed and selected for productivity and growth characteristics.
Example 1: Sequence selection and plasmid vectors The coding region of the gene for the hCG alpha polypeptide is shown in Fig 1. The coding region of the gene for the hCG alpha polypeptide sequence is referred to herein SEQ ID NO: 1.
The coding region of the gene for hCG beta polypeptide was used according to Fiddes and Goodman (1980). The sequence is banked as NP_000728 and is consistent with the protein sequences of CGbeta3, CGbeta5 and CGbeta7.
The sequence is referred to herein as SEQ ID NO: 2 The coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (a2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred to herein as SEQ ID NO: 3.
Two plasmids were used: the first, phCG, co-expresses hCG alpha and beta chains; and the second, expresses the sialyltransferase gene ST3GAL4.
Example 2a: Construction of the hCG expression vector phCG is a synthetic alpha chain of hCG, optimised for codon usage in mammalian cells including the native hCG alpha signal peptide. It may be engineered by methods well known in the art.
The coding sequence of hCG alpha polypeptide (SEQ ID NO: 1) and hCG
beta polypeptide (NP_000728, SEQ ID NO: 2) were amplified by PCR using methods well known in the art (see, for example, International Patent Application published as W02011/042688). The phCG alpha DNA was digested with BamHI
and Nhel and inserted into the sites BamHI and Nhel on a CMV driven mammalian expression vector (Crucell vector pcDNA3002Neo). This placed the gene in the correct orientation for expression driven by a CMVie promoter with a downstream BGH polyA signal. The native hCG beta gene including native signal peptide was digested with the restriction enzymes Ascl and Hpal and inserted into the Ascl and Hpal sites so that expression would be driven by a second CMVie promoter with an additional downstream BGH poly A signal. The vector backbone also included a neomycin resistance maker as well as elements required for selection and replication in prokaryotic cells. The vector was amplified and sequenced using general methods are known in the art. Sequences of the optimised hCG alpha and native (wild-type) beta chains are given in Figures 1 and 2, respectively.
All colonies selected for sequencing contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid phCG A+B was selected for transfection (Figure 4).
Example 2b: Construction of the ST3 expression vector The ST3GAL4 gene is expressed in pST3. The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4 (5T3, L23767, SEQ ID NO: 3, Fig 3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.
2,3STfw 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3' 2,3STrev 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3' The resulting amplified 5T3 DNA was digested with the restriction enzymes BamHI and MI I and inserted into the BamHI and MI I sites on the CMV
driven mammalian expression vector carrying a hygromycin resistance marker (vector pcDNA3002Neo) so that it is located downstream of CMVie promoter and upstream of BGH polyA sequence. The vector backbone also included a hygromycin resistance maker as well as elements required for selection and replication in prokaryotic cells. The vector was amplified as previously described and sequenced. Clone pST3#1 (Figure 5) contained the correct sequence according to SEQ ID NO: 3 and was selected for transfection.
Example 3 - Plasmid Transfection The plasmid phCG (Fig 4), which has a single Pvul site located in the prokaryotic beta-lactamase gene, was linearised with Pvul (New England Biolabs Cat. No. R0150). Linearized plasmid DNA was transfected into PER.C6 cells as follows.
Cell cultures were maintained in complete PERMAB medium (CD4PERMAB (Hyclone Cat. No. SH30871.01) supplemented with L-glutamine to 3mM final concentration (Invitrogen Cat. No. 25030-123) and Pluronic F68 at 1.0g/L final concentration (Invitrogen Cat. No. 24040-032) in 250m1 Erlenmeyer flasks. Cells were maintained in a shaking incubator (Kuhner Climo-shaker ISF1-X) set at 100rpm, 5% CO2 and 37 C, for at least 14 days prior to transfection.
hours prior to transfection, cells were transferred into fresh medium at a density of 0.5x106 cells/ml.
On the day of transfection, cells were counted in a Beckman Coulter ViCell XR to determine cell density and to ensure viability was >90%. Cells were harvested by centrifugation and resuspended in fresh PERMAB medium before being mixed with linearized phCG DNA. The cell/DNA mix was electro-shocked in the chamber of an electroporator set at 250V for 5msec, before being quickly transferred to 10m1 of pre-warmed PERMAB medium. This process was repeated a total of 6 times and all 6 transfections were pooled into a single T-175cm2 tissue culture flask. The flask was placed in a static incubator set at 37 C, 5% CO2.
After 48 hours, cells were resuspended in the appropriate volume of selective PERMAB
(complete PERMAB medium + G418 (125pg/mI)) to give a viable cell density of 0.5x106/ml.
The pool culture was passaged twice weekly, maintaining cells at a density of 0.3x106 cell/ml in selective medium until cell viability had increased to >50%. At this point, cells were transferred to shaking cultures in 250m1 Erlenmeyer flasks.
After several weeks in shaking culture, pool supernatant was sampled and assayed for hCG concentration. Once it was established that the pool was positive for hCG expression, cells were prepared for limited dilution cloning.
A cell suspension at 0.3 viable cells/ml was prepared in PERMAB medium supplemented with G-418 at 125pg/ml. The cell suspension was dispensed into 96 well flat bottomed tissue culture plates at 200p1/well and incubated in a humidified atmosphere at 37 C, 5% CO2 (Binder CB150). Plates were scanned regularly using the Genetix Clone Select Imager to track the growth of cells in each well.
After two weeks, 535 wells were identified that contained actively growing colonies of cells. Supernatants from these wells were sampled and assayed for hCG using a commercial kit (DRG diagnostics HCG ELISA Cat. No. EIA1469).
Based on the results from these assays, a total of 162 of the colonies were transferred into 24 well plates containing 0.5m1/well selective PERMAB medium.
When cells in the wells were near to confluency, supernatant from each of the wells was sampled and assayed for hCG levels. Based on these results, 91 of the best expressing cell lines were selected for expansion into T-25 flasks. These cell lines were again grown to near confluence, at which point supernatants were sampled and assayed for hCG as above. Based on these results, the 58 best expressing cell lines were expanded into T-75 flasks. Specific production rate (SPR) analysis was performed on each of these 58 cell lines by methods known in the art and specific productivity was expressed in pg/cell/day.
Thus, in this Example a plasmid incorporating hCG alpha and beta chains was transfected into PER.C60 cells and transfectants were selected using medium containing G418. Growing colonies of cells were screened for hCG concentration in the supernatant and those expressing the highest level were expanded further.
After subsequent rounds of expansion and screening, 20 clones that expressed hCG were selected for growth and productivity studies. Based on the results of these studies, 5 clones were selected for transfection with a second plasmid incorporating the ST3GAL4 gene.
Example 4 - Transfection with the ST3GAL4 plasmid pST3 Stable clones were generated as previously described in Example 3.
The five best clones produced by the method as described in Example 3 were selected for transfection with the ST3GAL4 plasmid pST3 (see Example 2, Figure 3 and 5). Transfection was performed by electroporation using the same method described in Example 3, except that transformants were selected in complete PERMAB medium supplemented with Hygromycin at 0.5ug/m1 instead of G418. Using this method, five pools were obtained that expressed hCG.
Samples of supernatants from the transfection pools were assed in a rat pharmacokinetic model and, based on this data along with data from the RCA-lectin binding assay (measuring exposed galactose on the hCG chains), a single pool was selected for further limited dilution cloning, by methods known in the art.
This dilution cloning yielded >600 clones each of which was also screened for hCG expression and the degree of exposed galactose as measured by RCA-lectin binding. Those clones with the lowest levels of exposed galactose were expanded further and samples were subjected to in vivo PK and corroborating lectin binding data. Five clones derived from the single pool were further assessed for growth and productivity characteristics.
Each of these clones was expanded and a seed stock cell bank was made following standard cryopreservation procedures. Stocks from the seed stock cell bank were thawed and found to be viable.
Example 5 - Analysis by isoelectric focussing.
Electrophoresis is defined as the transport of charged molecules through a solvent by an electrical field. The mobility of a biological molecule through an electric field will depend on the field strength, net charge on the molecule, size and shape of the molecule, ionic strength and properties of the medium through which the molecules migrate.
lsoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their pl. The pl is the pH at which a protein has no net charge and will not migrate in an electric field. The sialic acid content of the hCG
isoforms subtly alters the pl point for each isoform, which can be exploited using this technique to visualise the Per.C6 hCG isoforms from each clone.
The isoelectric points of the Per.C6 produced hCG isoforms were analyzed using isoelectric focussing. Per.C6 hCG was produced as described in Example 6.
Per.C6 hCG samples were separated on Novex IEF Gels containing 5%
polyacrylamide under native conditions on a pH 3.0 -7.0 gradient in an ampholyte solution pH 3.0 ¨ 7Ø Proteins were visualised using Coomassie Blue staining, using methods well known in the art.
Figure 6 shows the detection of rhCG lsoforms by IEF stained with Coomassie Blue in compositions produced according to the invention produced from the cloned cell lines made by the method set out in Examples 6 (Lanes 4, 5, 6 and 7, 15pg per lane); the CHO derived composition of the prior art, Ovitrelle (Lane 2, 15pg); and a human urinary product obtained from pregnant women Novarel (Lane 3, 15pg). The bands represent isoforms of hCG containing different numbers of sialic acid molecules. Figure 6 indicates that human cell line derived recombinant hCG engineered with a2,3- sialyltransferase (compositions according to the invention) is more acidic than Ovitrelle and urinary hCG from pregnant women.
Figure 7 compares the concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced using the cells of the invention (that is, using cell lines made by the method set out in Examples 1 to 4) which include the "Optimised"
alpha sub unit, compared with concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced in comparative example cells including the WT
(wild-type) alpha sub unit. Figure 7 shows that the cells of the invention express high amounts of hCG and low excess of the free beta subunit. In other words, the yield and purity of recombinant hCG produced by the cells and methods of the invention is markedly improved.
Table A indicates the percentage of free R-hCG Subunits in semi-purified samples of various batches of hCG produced using the cells of the invention (that is, using cell lines made by the method set out in Examples 1 to 4) which include the "Optimised" alpha sub unit ("Opt. alpha hCG"), compared with the percentage of free R-hCG Subunits in semi-purified samples of hCG produced in comparative example cells including the WT (wild-type) alpha sub unit ("WT alpha hCG"), as determined by Hydrophobic Phenyl-5PW HPLC chromatography.
Table A shows that the cells of the invention express high amounts of hCG
and a rather lower excess of the free beta subunit (5.7 ¨ 10.6%) compared to cells including the WT alpha sub unit (64 ¨ 66%). In other words, the yield and purity of recombinant hCG produced by the cells and methods of the invention is markedly improved.
Table A
% Free g-hCG
Clone (PHE - 5PW) WT alpha hCG (1G2) 64.1 WT alpha hCG (1G2) 66.0 Opt. alpha hCG
5.7 (13.8) Opt. alpha hCG (29) 7.5 Opt. alpha hCG (29) 6.6 Opt. alpha hCG (29) 10.6 Example 6 - Production and purification overview A procedure was developed to produce recombinant hCG in PER.C6 cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several hCG-producing PER.C6 cell lines.
Recombinant hCG from an a2,3-sialyltransferase transfected clone was prepared using the methods of Examples 1 to 4 described above.
The cells were grown in shaker flasks in 6GRO medium (SAFC) until a cell density of 1 x106 to 3x106 cells/ml was achieved. The cells were transferred to a 5L glass stirred tank bioreactor with a density of about 1 x106 cells/ml. The bioreactor worked in perfusion mode using a Proper1 media (Lonza).
Thereafter, purification of the product rhCG was carried out using various ultrafiltration steps, anion and cation exchange capture chromatography, hydrophobic chromatography and pseudo-affinity chromatography, by methods well known in the art.
During all chromatographic procedures, the presence of immunoreactive recombinant hCG was confirmed by ELISA (DRG EIA 1469) and IEF (Example 5).
Example 7 ¨ Sialic Acid Content Sialic acid is a protein-bound carbohydrate considered to be a mono-saccharide and occurs in combination with other mono- saccharides like galactose, mannose, glucosamine, galactosamine and fucose. The total sialic acid on purified rhCG according to the invention was measured using a method based on the method of Stanton et. al. (J. Biochem. Biophys. Methods. 30 (1995), 37 ¨ 48).
The total sialic acid content of samples of Per.C6 recombinant hCG
modified with a2,3- sialyltransferase (produced by the methods of Example 4 andf 6) were measured and the results are in Table 1 below [expressed in terms of a ratio of moles of sialic acid to moles of protein].
Example 8 ¨ hCG Bioassay according to USP
A hCG Bioassay was carried out, in order to determine the hCG specific activity, for each of the samples of Table 1 below. The activity was measured according to USP (USP Monographs: Chorionic Gonadotropin, USPC Official 8/1/09-11/30/09), using Ovitrelle as a standard. Ovitrelle has a biological activity of 26,000 IU/mg (Curr Med Res Opin. 2005 Dec; 21(12): 1969 ¨ 76). The acceptance limit was >21,000 IU hCG/mg. The biological activity for samples of human cell line derived hCG recombinant hCGs engineered with a2,3-sialyltransferase are shown in Table 1.
Table 1 Sample 1 2 3 4 5 6 Sialic acid (SA) content 13 13 13 13 13 15 (pmol SA/ pmolhCG) Potency Ill/mg 25363 25009 24904 24623 25645 26623 % Potency 97.5 96.2 95.8 94.7 98.6 102.4 (Ovitrelle 26000 IU/mg) Sample 7 8 9 10 11 Sialic acid (SA) content 14 14 13 13 13 (pmol SA/ pmolhCG) Potency IU/mg 27227 26142 26539 26181 24230 % Potency 104.7 100.5 102 101 93.2 (Ovitrelle 26000 Ill/mg) As seen above, the potency is similar to, and may be greater than, Ovitrelle (see e.g Sample 7).
References Andersen CY, Westergaard LG, and van Wely M. (2004). FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect? Reprod Biomed Online. 9(2), 231-236.
Bassett RM, and Driebergen R. (2005). Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH. Reprod Biomed Online.
10(2), 169-177.
D'Antonio M., Borrelli F. , Datola A., Bucci R. , Mascia M. , Polletta P., Piscitelli D., and Papoian R. (1999) Biological characterization of recombinant human follicle stimulating hormone isoforms. Human Reproduction 14, 1160-1167 Fiddes, J. C. and Goodman, H. M. (1979) Isolation, cloning and sequence analysis of the cDNA for the alpha-subunit of human chorionic gonadotropin. Nature, 281, 351-356.
Fiddes, J. C. and Goodman, H. M. (1980) The cDNA for the beta-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region. Nature, 286, 684-387.
Kagawa Y, Takasaki S, Utsumi J, Hosoi K, Shimizu H, Kochibe N, and Kobata A.
(1988).
Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells.
J Biol Chem. 263(33), 17508-17515.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1), 265-75.
Lowry, PJ, McLean, C, Jones RL and Satgunasingam N. (1976) Purification of anterior pituitary and hypothalamic hormones Clin Pathol Suppl (Assoc Clin Pathol). 7, 16-21.
Royle L, Radcliffe CM, Dwek RA and Rudd PM (2006) Methods in Molecular Biology, ed I
Brockhausen-Schutzbach (Humana Press), 347: Glycobiology protocols, 125-144.
Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 53(6), 604-616.
Svensson EC, Soreghan B, and Paulson JC. (1990) Organization of the beta-galactoside alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation. J Biol Chem. 265(34):20863-20868.
Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, and Kobata A
(1988).
Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells.
J Biol Chem. 263(8), 3657-3663.
Ulloa-Aguirre A, Midgley AR Jr, Beitins IZ, and Padmanabhan V. (1995).
Follicle-stimulating isohormones: characterization and physiological relevance. Endocr Rev.16(6), 765-787.
Ulloa-Aguirre A, Timossi C, Barrios-de-Tomasi J, Maldonado A, and Nayudu P.
(2003).
Impact of carbohydrate heterogeneity in function of follicle-stimulating hormone: studies derived from in vitro and in vivo models. Biol Reprod. 69(2), 379-389.
Optimised hCG alpha Nucleotide sequence of optimised hCG alpha (SEQ ID NO: 1) GTTCCTGCAC
GGAAAACCCC
CAGCAGAGCC
GACCAGCGAG
CTTCAAGGTG
Protein sequence of hCG optimised alpha QCMGCCFSRA
Human Chorionic Gonadotrophin beta polypeptide Accession number NP 000728 Nucleotide sequence of hCG beta (SEQ ID NO: 2) Nucleotide sequence GACATGGGCA
TGTGGAGAAG
CTGCCCCACC
CAACTACCGC
CCCCGTGGTC
CACTGACTGC
CTCCTCTTCC
CTCGGACACC
Protein sequence of hCG beta TTICAGYCPT
ALCRRSTTDC
Beta-galactoside alpha-2,3-sialyltransferase 4 Accession Number L23767 Nucleotide sequence of ST3GAL4 (SEQ ID NO: 3) CGTCATGGTG
CATCCCAGAG
CTTTGGCAAC
CAAGACGCCA
CCGGGTGCTA
CCGCTGTGTG
CAACAAGTAC
CGTGGGCTCC
AGTAGAAAAC
CTGGATTGAG
TCCCCTCATC
GATTGCAGCT
GCCCACCACG
TGCCGGCTTT
GATCACGCTC
TAAGCGGATG
Protein Sequence of ST3GAL4 ESKASKLFGN
IQSLRCRRCV
SAHFDPKVEN
LNPFFMEIAA
TIHYYEQITL
Optimised hCG alpha chain Nucleotide sequence of optimised hCG alpha chain (SEQ ID NO: 4) CTTCAGCCAG
CCCCACCCCC
CACCTGCTGC
GAACCACACC
Protein sequence of hCG optimised alpha chain KNVTSESTCC
hCG alpha polypeptide Accession number AH007338 Nucleotide sequence of hCG alpha (SEQ ID NO: 5) GTTTCTGCAT
GGAAAACCCA
CTCTAGAGCA
CACCTCAGAG
TTTCAAAGTG
Protein sequence of hCG alpha QCMGCCFSRA
PHARMACY, supra, at pages 780-820. The parenteral compositions can be supplied in liquid formulation or as a solid which will be mixed with a sterile injectable medium just prior to administration. In an embodiment, the parenteral compositions are supplied in dosage unit form for ease of administration and uniformity of dosage.
Detailed description of the invention The present invention will now be described in more detail with reference to the following Examples and to the attached drawings in which:
Figure 1 shows the nucleic acid and derived amino acid sequences of the optimised hCG alpha sequence;
Figure 2 shows the nucleic acid and derived amino acid sequences of hCG beta sequence;
Figure 3 shows the nucleic acid and derived amino acid sequences of ST3GAL4 Figure 4 shows a plasmid map of the phCGalpha/beta expression vector;
Figure 5 shows the a2,3-sialyltransferase (ST3GAL4) expression vector;
Figure 6 shows the detection of rhCG lsoforms by IEF stained with Coomassie Blue in compositions according to the invention (Lanes 4, 5, 6 and 7, 15 pg per lane); the CHO derived composition of the prior art, Ovitrelle (Lane 2, 15pg);
and a human urinary product obtained from pregnant women Novarel (Lane 3, 15pg);
and Figure 7 compares the concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced using the cells of the invention including the "Optimised" nucleic sequence of the alpha sub unit, compared with concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced in comparative example cells including the WT (wild-type) alpha sub unit;
Examples 1 to 7 Overview of cell line development process A single plasmid carrying hCG alpha and beta chains, each driven from a separate CMVie promoter, was transfected into PER.C60 cells by electroporation.
This plasmid carried a neomycin resistance gene cassette (Fig 4) allowing for selection of transfectants using G418 (Geneticin). Pools of transfectants were expanded and then subjected to limiting dilution cloning in 96 well plates under G418 selection. Clonal lines were expanded and high expressing colonies were identified based on hCG titre in the cell culture supernatant.
Five lead clones were selected, based on high productivity, and each was then transfected with a second plasmid expressing the a2,3-sialyltransferase gene ST3GAL4 along with the Hygromycin selection maker (Fig 5). Each of the five transfection pools was expanded and cell culture supernatants were assessed for hCG concentration and in vivo pharmacokinetics (PK).
Promising pools were then subjected to another round of limited dilution cloning. The resulting clones were expanded and cell culture supernatants were assessed for hCG concentration and exposed galactose on the hCG protein (by lectin ELISA). Those with a low level of exposed galactose were subjected to further assays including in vivo PK. Clones that produced hCG with optimum PK
profiles, had low exposed galactose and expressed the protein at high levels were assessed and selected for productivity and growth characteristics.
Example 1: Sequence selection and plasmid vectors The coding region of the gene for the hCG alpha polypeptide is shown in Fig 1. The coding region of the gene for the hCG alpha polypeptide sequence is referred to herein SEQ ID NO: 1.
The coding region of the gene for hCG beta polypeptide was used according to Fiddes and Goodman (1980). The sequence is banked as NP_000728 and is consistent with the protein sequences of CGbeta3, CGbeta5 and CGbeta7.
The sequence is referred to herein as SEQ ID NO: 2 The coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (a2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred to herein as SEQ ID NO: 3.
Two plasmids were used: the first, phCG, co-expresses hCG alpha and beta chains; and the second, expresses the sialyltransferase gene ST3GAL4.
Example 2a: Construction of the hCG expression vector phCG is a synthetic alpha chain of hCG, optimised for codon usage in mammalian cells including the native hCG alpha signal peptide. It may be engineered by methods well known in the art.
The coding sequence of hCG alpha polypeptide (SEQ ID NO: 1) and hCG
beta polypeptide (NP_000728, SEQ ID NO: 2) were amplified by PCR using methods well known in the art (see, for example, International Patent Application published as W02011/042688). The phCG alpha DNA was digested with BamHI
and Nhel and inserted into the sites BamHI and Nhel on a CMV driven mammalian expression vector (Crucell vector pcDNA3002Neo). This placed the gene in the correct orientation for expression driven by a CMVie promoter with a downstream BGH polyA signal. The native hCG beta gene including native signal peptide was digested with the restriction enzymes Ascl and Hpal and inserted into the Ascl and Hpal sites so that expression would be driven by a second CMVie promoter with an additional downstream BGH poly A signal. The vector backbone also included a neomycin resistance maker as well as elements required for selection and replication in prokaryotic cells. The vector was amplified and sequenced using general methods are known in the art. Sequences of the optimised hCG alpha and native (wild-type) beta chains are given in Figures 1 and 2, respectively.
All colonies selected for sequencing contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid phCG A+B was selected for transfection (Figure 4).
Example 2b: Construction of the ST3 expression vector The ST3GAL4 gene is expressed in pST3. The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4 (5T3, L23767, SEQ ID NO: 3, Fig 3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.
2,3STfw 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3' 2,3STrev 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3' The resulting amplified 5T3 DNA was digested with the restriction enzymes BamHI and MI I and inserted into the BamHI and MI I sites on the CMV
driven mammalian expression vector carrying a hygromycin resistance marker (vector pcDNA3002Neo) so that it is located downstream of CMVie promoter and upstream of BGH polyA sequence. The vector backbone also included a hygromycin resistance maker as well as elements required for selection and replication in prokaryotic cells. The vector was amplified as previously described and sequenced. Clone pST3#1 (Figure 5) contained the correct sequence according to SEQ ID NO: 3 and was selected for transfection.
Example 3 - Plasmid Transfection The plasmid phCG (Fig 4), which has a single Pvul site located in the prokaryotic beta-lactamase gene, was linearised with Pvul (New England Biolabs Cat. No. R0150). Linearized plasmid DNA was transfected into PER.C6 cells as follows.
Cell cultures were maintained in complete PERMAB medium (CD4PERMAB (Hyclone Cat. No. SH30871.01) supplemented with L-glutamine to 3mM final concentration (Invitrogen Cat. No. 25030-123) and Pluronic F68 at 1.0g/L final concentration (Invitrogen Cat. No. 24040-032) in 250m1 Erlenmeyer flasks. Cells were maintained in a shaking incubator (Kuhner Climo-shaker ISF1-X) set at 100rpm, 5% CO2 and 37 C, for at least 14 days prior to transfection.
hours prior to transfection, cells were transferred into fresh medium at a density of 0.5x106 cells/ml.
On the day of transfection, cells were counted in a Beckman Coulter ViCell XR to determine cell density and to ensure viability was >90%. Cells were harvested by centrifugation and resuspended in fresh PERMAB medium before being mixed with linearized phCG DNA. The cell/DNA mix was electro-shocked in the chamber of an electroporator set at 250V for 5msec, before being quickly transferred to 10m1 of pre-warmed PERMAB medium. This process was repeated a total of 6 times and all 6 transfections were pooled into a single T-175cm2 tissue culture flask. The flask was placed in a static incubator set at 37 C, 5% CO2.
After 48 hours, cells were resuspended in the appropriate volume of selective PERMAB
(complete PERMAB medium + G418 (125pg/mI)) to give a viable cell density of 0.5x106/ml.
The pool culture was passaged twice weekly, maintaining cells at a density of 0.3x106 cell/ml in selective medium until cell viability had increased to >50%. At this point, cells were transferred to shaking cultures in 250m1 Erlenmeyer flasks.
After several weeks in shaking culture, pool supernatant was sampled and assayed for hCG concentration. Once it was established that the pool was positive for hCG expression, cells were prepared for limited dilution cloning.
A cell suspension at 0.3 viable cells/ml was prepared in PERMAB medium supplemented with G-418 at 125pg/ml. The cell suspension was dispensed into 96 well flat bottomed tissue culture plates at 200p1/well and incubated in a humidified atmosphere at 37 C, 5% CO2 (Binder CB150). Plates were scanned regularly using the Genetix Clone Select Imager to track the growth of cells in each well.
After two weeks, 535 wells were identified that contained actively growing colonies of cells. Supernatants from these wells were sampled and assayed for hCG using a commercial kit (DRG diagnostics HCG ELISA Cat. No. EIA1469).
Based on the results from these assays, a total of 162 of the colonies were transferred into 24 well plates containing 0.5m1/well selective PERMAB medium.
When cells in the wells were near to confluency, supernatant from each of the wells was sampled and assayed for hCG levels. Based on these results, 91 of the best expressing cell lines were selected for expansion into T-25 flasks. These cell lines were again grown to near confluence, at which point supernatants were sampled and assayed for hCG as above. Based on these results, the 58 best expressing cell lines were expanded into T-75 flasks. Specific production rate (SPR) analysis was performed on each of these 58 cell lines by methods known in the art and specific productivity was expressed in pg/cell/day.
Thus, in this Example a plasmid incorporating hCG alpha and beta chains was transfected into PER.C60 cells and transfectants were selected using medium containing G418. Growing colonies of cells were screened for hCG concentration in the supernatant and those expressing the highest level were expanded further.
After subsequent rounds of expansion and screening, 20 clones that expressed hCG were selected for growth and productivity studies. Based on the results of these studies, 5 clones were selected for transfection with a second plasmid incorporating the ST3GAL4 gene.
Example 4 - Transfection with the ST3GAL4 plasmid pST3 Stable clones were generated as previously described in Example 3.
The five best clones produced by the method as described in Example 3 were selected for transfection with the ST3GAL4 plasmid pST3 (see Example 2, Figure 3 and 5). Transfection was performed by electroporation using the same method described in Example 3, except that transformants were selected in complete PERMAB medium supplemented with Hygromycin at 0.5ug/m1 instead of G418. Using this method, five pools were obtained that expressed hCG.
Samples of supernatants from the transfection pools were assed in a rat pharmacokinetic model and, based on this data along with data from the RCA-lectin binding assay (measuring exposed galactose on the hCG chains), a single pool was selected for further limited dilution cloning, by methods known in the art.
This dilution cloning yielded >600 clones each of which was also screened for hCG expression and the degree of exposed galactose as measured by RCA-lectin binding. Those clones with the lowest levels of exposed galactose were expanded further and samples were subjected to in vivo PK and corroborating lectin binding data. Five clones derived from the single pool were further assessed for growth and productivity characteristics.
Each of these clones was expanded and a seed stock cell bank was made following standard cryopreservation procedures. Stocks from the seed stock cell bank were thawed and found to be viable.
Example 5 - Analysis by isoelectric focussing.
Electrophoresis is defined as the transport of charged molecules through a solvent by an electrical field. The mobility of a biological molecule through an electric field will depend on the field strength, net charge on the molecule, size and shape of the molecule, ionic strength and properties of the medium through which the molecules migrate.
lsoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their pl. The pl is the pH at which a protein has no net charge and will not migrate in an electric field. The sialic acid content of the hCG
isoforms subtly alters the pl point for each isoform, which can be exploited using this technique to visualise the Per.C6 hCG isoforms from each clone.
The isoelectric points of the Per.C6 produced hCG isoforms were analyzed using isoelectric focussing. Per.C6 hCG was produced as described in Example 6.
Per.C6 hCG samples were separated on Novex IEF Gels containing 5%
polyacrylamide under native conditions on a pH 3.0 -7.0 gradient in an ampholyte solution pH 3.0 ¨ 7Ø Proteins were visualised using Coomassie Blue staining, using methods well known in the art.
Figure 6 shows the detection of rhCG lsoforms by IEF stained with Coomassie Blue in compositions produced according to the invention produced from the cloned cell lines made by the method set out in Examples 6 (Lanes 4, 5, 6 and 7, 15pg per lane); the CHO derived composition of the prior art, Ovitrelle (Lane 2, 15pg); and a human urinary product obtained from pregnant women Novarel (Lane 3, 15pg). The bands represent isoforms of hCG containing different numbers of sialic acid molecules. Figure 6 indicates that human cell line derived recombinant hCG engineered with a2,3- sialyltransferase (compositions according to the invention) is more acidic than Ovitrelle and urinary hCG from pregnant women.
Figure 7 compares the concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced using the cells of the invention (that is, using cell lines made by the method set out in Examples 1 to 4) which include the "Optimised"
alpha sub unit, compared with concentration of hCG ("alpha-beta hCG") and free beta chain ("beta hCG") produced in comparative example cells including the WT
(wild-type) alpha sub unit. Figure 7 shows that the cells of the invention express high amounts of hCG and low excess of the free beta subunit. In other words, the yield and purity of recombinant hCG produced by the cells and methods of the invention is markedly improved.
Table A indicates the percentage of free R-hCG Subunits in semi-purified samples of various batches of hCG produced using the cells of the invention (that is, using cell lines made by the method set out in Examples 1 to 4) which include the "Optimised" alpha sub unit ("Opt. alpha hCG"), compared with the percentage of free R-hCG Subunits in semi-purified samples of hCG produced in comparative example cells including the WT (wild-type) alpha sub unit ("WT alpha hCG"), as determined by Hydrophobic Phenyl-5PW HPLC chromatography.
Table A shows that the cells of the invention express high amounts of hCG
and a rather lower excess of the free beta subunit (5.7 ¨ 10.6%) compared to cells including the WT alpha sub unit (64 ¨ 66%). In other words, the yield and purity of recombinant hCG produced by the cells and methods of the invention is markedly improved.
Table A
% Free g-hCG
Clone (PHE - 5PW) WT alpha hCG (1G2) 64.1 WT alpha hCG (1G2) 66.0 Opt. alpha hCG
5.7 (13.8) Opt. alpha hCG (29) 7.5 Opt. alpha hCG (29) 6.6 Opt. alpha hCG (29) 10.6 Example 6 - Production and purification overview A procedure was developed to produce recombinant hCG in PER.C6 cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several hCG-producing PER.C6 cell lines.
Recombinant hCG from an a2,3-sialyltransferase transfected clone was prepared using the methods of Examples 1 to 4 described above.
The cells were grown in shaker flasks in 6GRO medium (SAFC) until a cell density of 1 x106 to 3x106 cells/ml was achieved. The cells were transferred to a 5L glass stirred tank bioreactor with a density of about 1 x106 cells/ml. The bioreactor worked in perfusion mode using a Proper1 media (Lonza).
Thereafter, purification of the product rhCG was carried out using various ultrafiltration steps, anion and cation exchange capture chromatography, hydrophobic chromatography and pseudo-affinity chromatography, by methods well known in the art.
During all chromatographic procedures, the presence of immunoreactive recombinant hCG was confirmed by ELISA (DRG EIA 1469) and IEF (Example 5).
Example 7 ¨ Sialic Acid Content Sialic acid is a protein-bound carbohydrate considered to be a mono-saccharide and occurs in combination with other mono- saccharides like galactose, mannose, glucosamine, galactosamine and fucose. The total sialic acid on purified rhCG according to the invention was measured using a method based on the method of Stanton et. al. (J. Biochem. Biophys. Methods. 30 (1995), 37 ¨ 48).
The total sialic acid content of samples of Per.C6 recombinant hCG
modified with a2,3- sialyltransferase (produced by the methods of Example 4 andf 6) were measured and the results are in Table 1 below [expressed in terms of a ratio of moles of sialic acid to moles of protein].
Example 8 ¨ hCG Bioassay according to USP
A hCG Bioassay was carried out, in order to determine the hCG specific activity, for each of the samples of Table 1 below. The activity was measured according to USP (USP Monographs: Chorionic Gonadotropin, USPC Official 8/1/09-11/30/09), using Ovitrelle as a standard. Ovitrelle has a biological activity of 26,000 IU/mg (Curr Med Res Opin. 2005 Dec; 21(12): 1969 ¨ 76). The acceptance limit was >21,000 IU hCG/mg. The biological activity for samples of human cell line derived hCG recombinant hCGs engineered with a2,3-sialyltransferase are shown in Table 1.
Table 1 Sample 1 2 3 4 5 6 Sialic acid (SA) content 13 13 13 13 13 15 (pmol SA/ pmolhCG) Potency Ill/mg 25363 25009 24904 24623 25645 26623 % Potency 97.5 96.2 95.8 94.7 98.6 102.4 (Ovitrelle 26000 IU/mg) Sample 7 8 9 10 11 Sialic acid (SA) content 14 14 13 13 13 (pmol SA/ pmolhCG) Potency IU/mg 27227 26142 26539 26181 24230 % Potency 104.7 100.5 102 101 93.2 (Ovitrelle 26000 Ill/mg) As seen above, the potency is similar to, and may be greater than, Ovitrelle (see e.g Sample 7).
References Andersen CY, Westergaard LG, and van Wely M. (2004). FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect? Reprod Biomed Online. 9(2), 231-236.
Bassett RM, and Driebergen R. (2005). Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH. Reprod Biomed Online.
10(2), 169-177.
D'Antonio M., Borrelli F. , Datola A., Bucci R. , Mascia M. , Polletta P., Piscitelli D., and Papoian R. (1999) Biological characterization of recombinant human follicle stimulating hormone isoforms. Human Reproduction 14, 1160-1167 Fiddes, J. C. and Goodman, H. M. (1979) Isolation, cloning and sequence analysis of the cDNA for the alpha-subunit of human chorionic gonadotropin. Nature, 281, 351-356.
Fiddes, J. C. and Goodman, H. M. (1980) The cDNA for the beta-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region. Nature, 286, 684-387.
Kagawa Y, Takasaki S, Utsumi J, Hosoi K, Shimizu H, Kochibe N, and Kobata A.
(1988).
Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells.
J Biol Chem. 263(33), 17508-17515.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1), 265-75.
Lowry, PJ, McLean, C, Jones RL and Satgunasingam N. (1976) Purification of anterior pituitary and hypothalamic hormones Clin Pathol Suppl (Assoc Clin Pathol). 7, 16-21.
Royle L, Radcliffe CM, Dwek RA and Rudd PM (2006) Methods in Molecular Biology, ed I
Brockhausen-Schutzbach (Humana Press), 347: Glycobiology protocols, 125-144.
Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 53(6), 604-616.
Svensson EC, Soreghan B, and Paulson JC. (1990) Organization of the beta-galactoside alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation. J Biol Chem. 265(34):20863-20868.
Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, and Kobata A
(1988).
Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells.
J Biol Chem. 263(8), 3657-3663.
Ulloa-Aguirre A, Midgley AR Jr, Beitins IZ, and Padmanabhan V. (1995).
Follicle-stimulating isohormones: characterization and physiological relevance. Endocr Rev.16(6), 765-787.
Ulloa-Aguirre A, Timossi C, Barrios-de-Tomasi J, Maldonado A, and Nayudu P.
(2003).
Impact of carbohydrate heterogeneity in function of follicle-stimulating hormone: studies derived from in vitro and in vivo models. Biol Reprod. 69(2), 379-389.
Optimised hCG alpha Nucleotide sequence of optimised hCG alpha (SEQ ID NO: 1) GTTCCTGCAC
GGAAAACCCC
CAGCAGAGCC
GACCAGCGAG
CTTCAAGGTG
Protein sequence of hCG optimised alpha QCMGCCFSRA
Human Chorionic Gonadotrophin beta polypeptide Accession number NP 000728 Nucleotide sequence of hCG beta (SEQ ID NO: 2) Nucleotide sequence GACATGGGCA
TGTGGAGAAG
CTGCCCCACC
CAACTACCGC
CCCCGTGGTC
CACTGACTGC
CTCCTCTTCC
CTCGGACACC
Protein sequence of hCG beta TTICAGYCPT
ALCRRSTTDC
Beta-galactoside alpha-2,3-sialyltransferase 4 Accession Number L23767 Nucleotide sequence of ST3GAL4 (SEQ ID NO: 3) CGTCATGGTG
CATCCCAGAG
CTTTGGCAAC
CAAGACGCCA
CCGGGTGCTA
CCGCTGTGTG
CAACAAGTAC
CGTGGGCTCC
AGTAGAAAAC
CTGGATTGAG
TCCCCTCATC
GATTGCAGCT
GCCCACCACG
TGCCGGCTTT
GATCACGCTC
TAAGCGGATG
Protein Sequence of ST3GAL4 ESKASKLFGN
IQSLRCRRCV
SAHFDPKVEN
LNPFFMEIAA
TIHYYEQITL
Optimised hCG alpha chain Nucleotide sequence of optimised hCG alpha chain (SEQ ID NO: 4) CTTCAGCCAG
CCCCACCCCC
CACCTGCTGC
GAACCACACC
Protein sequence of hCG optimised alpha chain KNVTSESTCC
hCG alpha polypeptide Accession number AH007338 Nucleotide sequence of hCG alpha (SEQ ID NO: 5) GTTTCTGCAT
GGAAAACCCA
CTCTAGAGCA
CACCTCAGAG
TTTCAAAGTG
Protein sequence of hCG alpha QCMGCCFSRA
Claims (12)
1. A polynucleotide sequence comprising a nucleic acid sequence according to SEQ ID NO: 1.
2. A polynucleotide sequence comprising a nucleic acid sequence according to SEQ ID NO: 4.
3. A polynucleotide sequence comprising a nucleic acid sequence with at least 97% sequence identity with the nucleic acid of SEQ ID NO: 1.
4. A polynucleotide sequence comprising a nucleic acid sequence with at least 97% sequence identity with the nucleic acid of SEQ ID NO: 4.
5. A cell characterized in that it comprises integrated into its genome a sequence coding for the a chain of hCG selected from: a sequence according to SEQ ID NO: 1; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO:1; a sequence which has at least 97 % homology with the sequence of SEQ ID NO:1; a sequence according to SEQ ID NO. 4; a sequence which has at least 96.5 % homology with the sequence of SEQ ID NO:
4; and a sequence which has at least 97 % homology with the sequence of SEQ
ID NO:4.
4; and a sequence which has at least 97 % homology with the sequence of SEQ
ID NO:4.
6. A cell according to claim 5 which is a PER.C6 ® cell such as a PER.C6 ®
cell deposited under ECACC no. 96022940.
cell deposited under ECACC no. 96022940.
7. A PER.C6 ® cell such as a PER.C6 ® cell deposited under ECACC
no.
96022940, characterized in that it further comprises integrated into its genome a sequence coding for the a chain of hCG.
no.
96022940, characterized in that it further comprises integrated into its genome a sequence coding for the a chain of hCG.
8. A cell according to any of claims 5 to 7 characterized in that it further comprises integrated into its genome a cDNA encoding alpha-2,3-sialyltransferase .
9. A cell according to any of claims 5 to 8 characterized in that it further comprises integrated into its genome a nucleic acid sequence coding for the .beta.
chain of hCG.
chain of hCG.
10. A method for producing recombinant protein in a cell, comprising culturing a cell according to any of claims 5 to 9 in a suitable medium and harvesting the recombinant protein from said cell and/or said medium.
11. A method for producing recombinant hCG in a cell, comprising culturing the cell in a suitable medium and harvesting the recombinant protein (hCG) from said cell and/or said medium, wherein the cell is a PER.C6 ® cell comprising integrated into its genome a sequence coding for the a chain of hCG.
12. A recombinant hCG ("rhCG" or "rechCG") which includes .alpha.2,3- and .alpha.2,6-sialylation, wherein the recombinant hCG is produced in a cell according to any of claims 5 to 9.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP15164965.4 | 2015-04-24 | ||
EP15164965 | 2015-04-24 | ||
PCT/EP2016/059006 WO2016170113A1 (en) | 2015-04-24 | 2016-04-22 | Method of production of gonadotrophin |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2975449A1 true CA2975449A1 (en) | 2016-10-27 |
Family
ID=53002568
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2975449A Pending CA2975449A1 (en) | 2015-04-24 | 2016-04-22 | Method of production of gonadotrophin |
Country Status (32)
Country | Link |
---|---|
US (1) | US10464984B2 (en) |
EP (1) | EP3286209B1 (en) |
JP (1) | JP6964519B2 (en) |
KR (1) | KR102658666B1 (en) |
CN (2) | CN114107315A (en) |
AU (1) | AU2016251314B2 (en) |
BR (1) | BR112017016895A2 (en) |
CA (1) | CA2975449A1 (en) |
CL (1) | CL2017002661A1 (en) |
CO (1) | CO2017011964A2 (en) |
DK (1) | DK3286209T3 (en) |
EA (1) | EA034022B1 (en) |
ES (1) | ES2837111T3 (en) |
HK (1) | HK1246325A1 (en) |
HR (1) | HRP20201945T1 (en) |
HU (1) | HUE051879T2 (en) |
IL (1) | IL253694B (en) |
LT (1) | LT3286209T (en) |
MA (1) | MA41949B1 (en) |
MD (1) | MD3286209T2 (en) |
MX (1) | MX2017010884A (en) |
MY (1) | MY185823A (en) |
PH (1) | PH12017501585A1 (en) |
PT (1) | PT3286209T (en) |
RS (1) | RS61163B1 (en) |
SA (1) | SA517390029B1 (en) |
SG (1) | SG11201706832PA (en) |
SI (1) | SI3286209T1 (en) |
TN (1) | TN2017000328A1 (en) |
UA (1) | UA124187C2 (en) |
WO (1) | WO2016170113A1 (en) |
ZA (1) | ZA201707136B (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AR112541A1 (en) | 2017-09-01 | 2019-11-06 | Ferring Bv | COMPOSITION FOR CONTROLLED OVARIAN STIMULATION WITH RECOMBINANT HEF |
CN108264549A (en) * | 2018-03-29 | 2018-07-10 | 北京伟杰信生物科技有限公司 | Recombinate the preparation method and application of chorionic gonadotrophin rhCG |
US20210038694A1 (en) | 2018-04-30 | 2021-02-11 | Ferring B.V. | Composition for controlled ovarian stimulation |
CN110812366B (en) * | 2019-11-18 | 2023-11-17 | 珠海丽凡达生物技术有限公司 | mRNA medicine for hormone supplement and preparation method thereof |
CN113337562B (en) * | 2021-05-24 | 2022-06-03 | 宁波人健药业集团股份有限公司 | Method for producing rhCG by using CHO (Chinese hamster ovary) cells through high-efficiency fermentation |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6855544B1 (en) | 1999-04-15 | 2005-02-15 | Crucell Holland B.V. | Recombinant protein production in a human cell |
AU2001231531A1 (en) * | 2000-02-11 | 2001-08-20 | Maxygen Aps | Follicle stimulating hormones |
US20030036181A1 (en) | 2000-06-30 | 2003-02-20 | Okkels Jens Sigurd | Peptide extended glycosylated polypeptides |
CN1194016C (en) * | 2000-12-29 | 2005-03-23 | 申庆祥 | New human chorionic gonadotropin-lutropin fusion protein and its prepn and use |
CN101250531B (en) * | 2006-11-27 | 2013-04-24 | 株式会社Lg生命科学 | Nucleotide sequence, expression vector containing the same, zooblast inverted from said vector and method for producing human FSH using the zooblast |
TWI532495B (en) | 2009-10-05 | 2016-05-11 | 菲瑞茵國際中心股份有限公司 | Pharmaceutical preparation |
US8326547B2 (en) * | 2009-10-07 | 2012-12-04 | Nanjingjinsirui Science & Technology Biology Corp. | Method of sequence optimization for improved recombinant protein expression using a particle swarm optimization algorithm |
WO2011084145A2 (en) * | 2009-12-21 | 2011-07-14 | Pharmathene, Inc. | Recombinant butyrylcholinesterases and truncates thereof |
WO2013151670A2 (en) | 2012-04-02 | 2013-10-10 | modeRNA Therapeutics | Modified polynucleotides for the production of nuclear proteins |
JP2015518705A (en) * | 2012-04-02 | 2015-07-06 | モデルナ セラピューティクス インコーポレイテッドModerna Therapeutics,Inc. | Modified polynucleotides for the production of biologics and proteins associated with human diseases |
CN103539862B (en) * | 2013-11-01 | 2015-04-01 | 广州联康生物科技有限公司 | Long-acting recombinant follicle-stimulating hormone and application thereof |
-
2016
- 2016-04-22 SG SG11201706832PA patent/SG11201706832PA/en unknown
- 2016-04-22 HU HUE16721722A patent/HUE051879T2/en unknown
- 2016-04-22 CA CA2975449A patent/CA2975449A1/en active Pending
- 2016-04-22 BR BR112017016895A patent/BR112017016895A2/en active Search and Examination
- 2016-04-22 US US15/566,961 patent/US10464984B2/en active Active
- 2016-04-22 MY MYPI2017703981A patent/MY185823A/en unknown
- 2016-04-22 ES ES16721722T patent/ES2837111T3/en active Active
- 2016-04-22 UA UAA201711487A patent/UA124187C2/en unknown
- 2016-04-22 AU AU2016251314A patent/AU2016251314B2/en active Active
- 2016-04-22 SI SI201631009T patent/SI3286209T1/en unknown
- 2016-04-22 DK DK16721722.3T patent/DK3286209T3/en active
- 2016-04-22 MA MA41949A patent/MA41949B1/en unknown
- 2016-04-22 LT LTEP16721722.3T patent/LT3286209T/en unknown
- 2016-04-22 MX MX2017010884A patent/MX2017010884A/en unknown
- 2016-04-22 TN TNP/2017/000328A patent/TN2017000328A1/en unknown
- 2016-04-22 WO PCT/EP2016/059006 patent/WO2016170113A1/en active Application Filing
- 2016-04-22 EP EP16721722.3A patent/EP3286209B1/en active Active
- 2016-04-22 CN CN202111399800.1A patent/CN114107315A/en active Pending
- 2016-04-22 CN CN201680022637.8A patent/CN107532172A/en active Pending
- 2016-04-22 JP JP2017555503A patent/JP6964519B2/en active Active
- 2016-04-22 PT PT167217223T patent/PT3286209T/en unknown
- 2016-04-22 KR KR1020177022423A patent/KR102658666B1/en active IP Right Grant
- 2016-04-22 RS RS20201492A patent/RS61163B1/en unknown
- 2016-04-22 MD MDE20180206T patent/MD3286209T2/en unknown
- 2016-04-22 EA EA201791724A patent/EA034022B1/en unknown
-
2017
- 2017-07-27 IL IL253694A patent/IL253694B/en unknown
- 2017-09-04 PH PH12017501585A patent/PH12017501585A1/en unknown
- 2017-09-26 SA SA517390029A patent/SA517390029B1/en unknown
- 2017-10-19 CL CL2017002661A patent/CL2017002661A1/en unknown
- 2017-10-20 ZA ZA2017/07136A patent/ZA201707136B/en unknown
- 2017-11-24 CO CONC2017/0011964A patent/CO2017011964A2/en unknown
-
2018
- 2018-05-08 HK HK18105909.9A patent/HK1246325A1/en unknown
-
2020
- 2020-12-04 HR HRP20201945TT patent/HRP20201945T1/en unknown
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
RU2745557C1 (en) | Recombinant follicle-stimulating hormone (FSH), including alpha-2,3 - and alpha-2,6-sialation | |
JP6718951B2 (en) | Compositions for regulated ovarian stimulation | |
JP7292153B2 (en) | Pharmaceutical formulation | |
US10464984B2 (en) | Optimized nucleic acid sequences coding for the alpha-chain of human chorionic gonadotropin | |
EP2717904A1 (en) | Pharmaceutical preparation comprising recombinant fsh | |
Class et al. | Patent application title: Pharmaceutical Preparation Comprising Recombinant HcG Inventors: Ian Cottingham (St. Prex, CH) Daniel Plaksin (St. Prex, CH) Richard Boyd White (San Diego, CA, US) Assignees: Ferring BV | |
TW201302783A (en) | Pharmaceutical preparation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20210401 |
|
EEER | Examination request |
Effective date: 20210401 |
|
EEER | Examination request |
Effective date: 20210401 |
|
EEER | Examination request |
Effective date: 20210401 |
|
FZDE | Discontinued |
Effective date: 20230918 |
|
FZDC | Discontinued application reinstated |
Effective date: 20240426 |
|
FZDC | Discontinued application reinstated |
Effective date: 20240426 |